Dna polymerases and related methods

ABSTRACT

Disclosed are mutant DNA polymerases having improved extension rates relative to a corresponding, unmodified polymerase. The mutant polymerases are useful in a variety of disclosed primer extension methods. Also disclosed are related compositions, including recombinant nucleic acids, vectors, and host cells, which are useful, e.g., for production of the mutant DNA polymerases.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.14/601,168, filed on Jan. 20, 2015, which is a divisional of U.S.application Ser. No. 11/873,896, filed on Oct. 17, 2007, now U.S. Pat.No. 8,962,293, which claims the benefit of priority to U.S. ProvisionalApplication No. 60/852,882, filed on Oct. 18, 2006, the entire contentsof each of which are hereby incorporated by reference in their entirety.

REFERENCE TO A “SEQUENCE LISTING,” A TABLE, OR A COMPUTER PROGRAMLISTING APPENDIX SUBMITTED AS AN ASCII TEXT FILE

The Sequence Listing written in file-501-1.TXT, created on Jan. 12,2011, 156,000 bytes, machine format IBM-PC, MS-Windows operating system,is hereby incorporated by reference in its entirety for all purposes.

FIELD OF INVENTION

The present invention lies in the field of DNA polymerases and their usein various applications, including nucleic acid primer extension andamplification.

BACKGROUND OF THE INVENTION

DNA polymerases are responsible for the replication and maintenance ofthe genome, a role that is central to accurately transmitting geneticinformation from generation to generation. DNA polymerases function incells as the enzymes responsible for the synthesis of DNA. Theypolymerize deoxyribonucleoside triphosphates in the presence of a metalactivator, such as Mg²⁺, in an order dictated by the DNA template orpolynucleotide template that is copied. In vivo, DNA polymerasesparticipate in a spectrum of DNA synthetic processes including DNAreplication, DNA repair, recombination, and gene amplification. Duringeach DNA synthetic process, the DNA template is copied once or at most afew times to produce identical replicas. In contrast, in vitro, DNAreplication can be repeated many times such as, for example, duringpolymerase chain reaction (see, e.g., U.S. Pat. No. 4,683,202 toMullis).

In the initial studies with polymerase chain reaction (PCR), the DNApolymerase was added at the start of each round of DNA replication (seeU.S. Pat. No. 4,683,202, supra). Subsequently, it was determined thatthermostable DNA polymerases could be obtained from bacteria that growat elevated temperatures, and that these enzymes need to be added onlyonce (see U.S. Pat. No. 4,889,818 to Gelfand and U.S. Pat. No. 4,965,188to Mullis). At the elevated temperatures used during PCR, these enzymesare not irreversibly inactivated. As a result, one can carry outrepetitive cycles of polymerase chain reactions without adding freshenzymes at the start of each synthetic addition process. DNApolymerases, particularly thermostable polymerases, are the key to alarge number of techniques in recombinant DNA studies and in medicaldiagnosis of disease. For diagnostic applications in particular, atarget nucleic acid sequence may be only a small portion of the DNA orRNA in question, so it may be difficult to detect the presence of atarget nucleic acid sequence without amplification. Due to theimportance of DNA polymerases in biotechnology and medicine, it would behighly advantageous to generate DNA polymerase mutants having desiredenzymatic properties such as, for example, improved primer extensionrates, reverse transcription efficiency, or amplification ability.

The overall folding pattern of polymerases resembles the human righthand and contains three distinct subdomains of palm, fingers, and thumb.(See Beese et al., Science 260:352-355, 1993); Patel et al.,Biochemistry 34:5351-5363, 1995). While the structure of the fingers andthumb subdomains vary greatly between polymerases that differ in sizeand in cellular functions, the catalytic palm subdomains are allsuperimposable. For example, motif A, which interacts with the incomingdNTP and stabilizes the transition state during chemical catalysis, issuperimposable with a mean deviation of about one A amongst mammalianpol a and prokaryotic pol I family DNA polymerases (Wang et al., Cell89:1087-1099, 1997). Motif A begins structurally at an antiparallelβ-strand containing predominantly hydrophobic residues and continues toan α-helix. The primary amino acid sequence of DNA polymerase activesites is exceptionally conserved. In the case of motif A, for example,the sequence DYSQIELR (SEQ ID NO:22) is retained in polymerases fromorganisms separated by many millions years of evolution, including,e.g., Thermus aquaticus, Chlamydia trachomatis, and Escherichia coli.Taken together, these observations indicate that polymerases function bysimilar catalytic mechanisms.

In addition to being well-conserved, the active site of DNA polymeraseshas also been shown to be relatively mutable, capable of accommodatingcertain amino acid substitutions without reducing DNA polymeraseactivity significantly. (See, e.g., U.S. Pat. No. 6,602,695 to Patel etal.) Such mutant DNA polymerases can offer various selective advantagesin, e.g., diagnostic and research applications comprising nucleic acidsynthesis reactions. Thus, there is a need in the art for identificationof amino acid positions amenable to mutation to yield improvedpolymerase activity, including, for example, improved extension rates,reverse transcription efficiency, or amplification ability. The presentinvention, as set forth herein, meets these and other needs.

BRIEF SUMMARY OF THE INVENTION

The present invention provides DNA polymerases having improved enzymeactivity relative to the corresponding unmodified polymerase and whichare useful in a variety of nucleic acid synthesis applications. In someembodiments, the polymerase comprises an amino acid sequence having atleast one of the following motifs in the polymerase domain:

a)X_(a1)-X_(a2)-X_(a3)-X_(a4)-R-X_(a6)-X_(a7)-X_(a8)-K-L-X_(a11)-X_(a12)-T-Y-X_(a15)-X_(a16)(SEQ ID NO:1);

wherein X_(a1) is I or L;

X_(a2) is L or Q;

X_(a3) is Q, H or E;

X_(a4) is Y, H or F;

X_(a6) is E, Q or K;

X_(a7) is I, L or Y;

X_(a8) is an amino acid other than Q, T, M, G or L;

X_(a11) is K or Q;

X_(a12) is S or N;

X_(a15) is I or V; and

X_(a16) is E or D;

b) T-G-R-L-S—S-X_(b7)-X_(b8)-P-N-L-Q-N (SEQ ID NO:2); wherein

X_(b7) is S or T; and

X_(b8) is an amino acid other than D, E or N; and

c) X_(c1)-X_(c2)-X_(c3)-X_(c4)-X_(c5)-X_(c6)-X_(c7)-D-Y-S-Q-I-E-L-R (SEQID NO:3); wherein

X_(c1) is G, N, or D;

X_(c2) is W or H;

X_(c3) is W, A, L, or V;

X_(c4) is an amino acid other than I or L;

X_(c5) is V, F or L;

X_(c6) is an amino acid other than S, A, V, or G; and

X_(c7) is A or L,

wherein the polymerase has an improved nucleic acid extension rateand/or an improved reverse transcription efficiency relative to anotherwise identical polymerase wherein X_(a8) is an amino acid selectedfrom Q, T, M, G or L; X_(b8) is an amino acid selected from D, E or Nand/or X_(c6) is an amino acid selected from S, A, V, or G (i.e., areference polymerase). In some embodiments of the reference polymerase(e.g., Z05 or CS5/CS6), X_(a8) is Q, T, M, G or L, X_(b8) is D, E or N,X_(c4) is I or L, and X_(c6) is S, A, V, or G (SEQ ID NOS:23 and 24). Insome embodiments of the reference polymerase, X_(b8) is D, E or N (SEQID NOS:25 and 26).

With respect to motif a)X_(a1)-X_(a2)-X_(a3)-X_(a4)-R—X_(a6)-X_(a7)-X_(a8)-K-L-X_(a11)-X_(a12)-T-Y-X_(a15)-X_(a16)(SEQ ID NO:1), in some embodiments, X_(a8) is a D- or L-amino acidselected from the group consisting of: A, C, D, E, F, H, I, K, N, P, R,S, V, W, Y (SEQ ID NO:27), and analogs thereof. In some embodiments,X_(a8) is an amino acid selected from the group consisting of: R, K andN (SEQ ID NO:28). In some embodiments, X_(a8) is Arginine (R) (SEQ IDNO:29).

With respect to motif b) T-G-R-L-S—S-X_(b7)-X_(b8)-P-N-L-Q-N (SEQ IDNO:2), in some embodiments, X_(b8) is D- or L-amino acid selected fromthe group consisting of: A, C, F, G, H, I, K, L, M, P, Q, R, S, T, V, W,Y (SEQ ID NO:30), and analogs thereof. In some embodiments, X_(b8) is anamino acid selected from the group consisting of: G, A, S, T, R, K, Q,L, V and I (SEQ ID NO:31). In some embodiments, X_(b8) is an amino acidselected from the group consisting of: G, T, R, K and L (SEQ ID NO:32).

With respect to motif c)X_(c1)-X_(c2)-X_(c3)-X_(c4)-X_(c5)-X_(c6)-X_(c7)-D-Y-S-Q-I-E-L-R (SEQ IDNO:3), in some embodiments, X_(c4) is a D- or L-amino acid selected fromthe group consisting of: A, C, D, E, F, G, H, K, M, N, P, Q, R, S, T, V,W, Y (SEQ ID NO:33), and analogs thereof. In some embodiments, X_(c4) isan amino acid selected from the group consisting of F and Y (SEQ IDNO:34). In some embodiments, X_(c4) is phenylalanine (F) (SEQ ID NO:35).In some embodiments, X_(c6) is an amino acid selected from the groupconsisting of C, D, E, F, H, I, K, L, M, N, P, Q, R, T, W and Y (SEQ IDNO:36). In some embodiments, X_(c6) is an amino acid selected from thegroup consisting of F and Y (SEQ ID NO:37). In some embodiments, X_(c6)is phenylalanine (F) (SEQ ID NO:38).

In some embodiments, the improved polymerases (e.g., Z05 or CS5/CS6)that comprise at least one of Arginine (R) at position X_(a8); Glycine(G) at position X_(b8); Phenylalanine (F) at position X_(c4); and/orPhenylalanine (F) at position X_(c6) (SEQ ID NOS:39-68).

In some embodiments, the DNA polymerases of the invention are modifiedversions of an unmodified polymerase. In its unmodified form, thepolymerase includes an amino acid sequence having the following motifsin the polymerase domain:

-   -   X_(a1)-X_(a2)-X_(a3)-X_(a4)-R—X_(a6)-X_(a7)-X_(a8)-K-L-X_(a11)-X_(a12)-T-Y-X_(a15)-X_(a16)        (SEQ ID NO:69); wherein X_(a1) is I or L; X_(a2) is L or Q;        X_(a3) is Q, H or E; X_(a4) is Y, H or F; X_(a6) is E, Q or K;        X_(a7) is I, L or Y; X_(a8) is Q, T, M, G or L; X_(a11) is K or        Q; X_(a12) is S or N; X_(a15) is I or V; and X_(a16) is E or D;    -   T-G-R-L-S-S-X_(b7)-X_(b8)-P-N-L-Q-N (SEQ ID NO:70); wherein        X_(b7) is S or T; and X_(b8) is D, E or N; and    -   X_(c1)-X_(c2)-X_(c3)-X_(c4)-X_(c5)-X_(c6)-X_(c7)-D-Y-S-Q-I-E-L-R        (SEQ ID NO:71); wherein X_(c1) is G, N or D; X_(c2) is W or H;        X_(c3) is W, A, L or V; X_(c4) is I or L; X_(c5) is V, F or L;        X_(c6) is S, A, V or G; and X_(c7) is A or L.

Various DNA polymerases are amenable to mutation according to thepresent invention. Particularly suitable are thermostable polymerases,including wild-type or naturally occurring thermostable polymerases fromvarious species of thermophilic bacteria, as well as thermostablepolymerases derived from such wild-type or naturally occurring enzymesby amino acid substitution, insertion, or deletion, or othermodification. Exemplary unmodified forms of polymerase include, e.g.,CS5, CS6 or Z05 DNA polymerase, or a functional DNA polymerase having atleast 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequenceidentity thereto. Other unmodified polymerases include, e.g., DNApolymerases from any of the following species of thermophilic bacteria(or a functional DNA polymerase having at least 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98% or 99% sequence identity to such a polymerase):Thermotoga maritima; Thermus aquaticus; Thermus thermophilus; Thermusflavus; Thermus filiformis; Thermus sp. sps17; Thermus sp. Z05;Thermotoga neopolitana; Thermosipho africanus; Thermus caldophilus orBacillus caldotenax. Suitable polymerases also include those havingreverse transcriptase (RT) activity and/or the ability to incorporateunconventional nucleotides, such as ribonucleotides or other 2′-modifiednucleotides.

In some embodiments, the unmodified form of the polymerase comprises achimeric polymerase. In one embodiment, for example, the unmodified formof the chimeric polymerase is CS5 DNA polymerase (SEQ ID NO:18), CS6 DNApolymerase (SEQ ID NO:19), or a polymerase having at least 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the CS5DNA polymerase or the CS6 DNA polymerase. In specific variations, theunmodified form of the chimeric polymerase includes one or more aminoacid substitutions relative to SEQ ID NO:18 or SEQ ID NO:19 that areselected from G46E, L329A, and E678G. For example, the unmodified formof the mutant or improved polymerase can be G46E CS5; G46E L329A CS5;G46E E678G CS5; or G46E L329A E678G CS5. In exemplary embodiments, theseunmodified forms are substituted to provide a mutant polymeraseincluding one or more amino acid substitutions selected from S671F,D640G, Q601R, and I669F. For example, the mutant or improved DNApolymerase can be any one of the following: G46E S671F CS5; G46E D640GCS5; G46E Q601R CS5; G46E I669F CS5; G46E D640G S671F CS5; G46E L329AS671F CS5; G46E L329A D640G CS5; G46E L329A Q601R CS5; G46E L329A I669FCS5; G46E L329A D640G S671F CS5; G46E S671F E678G CS5; G46E D640G E678GCS5; G46E Q601R E678G CS5; G46E I669F E678G CS5; G46E L329A S671F E678GCS5; G46E L329A D640G E678G CS5; G46E L329A Q601R E678G CS5; G46E L329AQ601R D640G I669F S671F E678G CS5; G46E L329A I669F E678G CS5; or thelike.

In some embodiments, the polymerase is a CS5 polymerase (SEQ ID NO:15),a CS6 polymerase (SEQ ID NO:16) or a Z05 polymerase (SEQ ID NO:6),wherein X_(b8) is an amino acid selected from the group consisting of:G, T, R, K and L. For example, the CS5 or CS6 polymerase can be selectedfrom the following: D640G, D640T, D640R, D640K and D640L. The Z05polymerase can be selected from the group consisting of: D580G, D580T,D580R, D580K and D580L.

The mutant or improved polymerase can include other, non-substitutionalmodifications. One such modification is a thermally reversible covalentmodification that inactivates the enzyme, but which is reversed toactivate the enzyme upon incubation at an elevated temperature, such asa temperature typically used for primer extension. In one embodiment,the mutant or improved polymerase comprising the thermally reversiblecovalent modification is produced by a reaction, carried out at alkalinepH at a temperature that is less than about 25° C., of a mixture of athermostable DNA polymerase and a dicarboxylic acid anhydride having oneof the following formulas I or II:

wherein R₁ and R₂ are hydrogen or organic radicals, which may be linked;or

wherein R₁ and R₂ are organic radicals, which may linked, and thehydrogens are cis. In a specific variation of such an enzyme, theunmodified form of the polymerase is G64E CS5.

In some embodiments, the extension rate is determined using asingle-stranded DNA as a template (e.g, M13mp18, HIV), primed with anappropriate primer (e.g., a polynucleotide of the nucleic acid sequence5′-GGGAAGGGCGATCGGTGCGGGCCTCTTCGC-3′ (SEQ ID NO:72)), and detectingformation of double-stranded DNA by measuring the incorporation of afluorophore at regular time intervals (e.g., every 5, 10, 15, 20, 30 or60 seconds), as described herein. The extension rate of a polymerase ofthe invention can be compared to the extension rate of a referencepolymerase (e.g., a naturally occurring or unmodified polymerase), overa preselected unit of time, as described herein.

In various other aspects, the present invention provides a recombinantnucleic acid encoding a mutant or improved DNA polymerase as describedherein, a vector comprising the recombinant nucleic acid, and a hostcell transformed with the vector. In certain embodiments, the vector isan expression vector. Host cells comprising such expression vectors areuseful in methods of the invention for producing the mutant or improvedpolymerase by culturing the host cells under conditions suitable forexpression of the recombinant nucleic acid. The polymerases of theinvention may be contained in reaction mixtures and/or kits. Theembodiments of the recombinant nucleic acids, host cells, vectors,expression vectors, reaction mixtures and kits are as described aboveand herein.

In yet another aspect, a method for conducting primer extension isprovided. The method generally includes contacting a mutant or improvedDNA polymerase of the invention with a primer, a polynucleotidetemplate, and free nucleotides under conditions suitable for extensionof the primer, thereby producing an extended primer. The polynucleotidetemplate can be, for example, an RNA or DNA template. The freenucleotides can include unconventional nucleotides such as, e.g.,ribonucleotides and/or labeled nucleotides. Further, the primer and/ortemplate can include one or more nucleotide analogs. In some variations,the primer extension method is a method for polynucleotide amplificationthat includes contacting the mutant or improved DNA polymerase with aprimer pair, the polynucleotide template, and the free nucleotides underconditions suitable for amplification of the polynucleotide.

The present invention also provides a kit useful in such a primerextension method. Generally, the kit includes at least one containerproviding a mutant or improved DNA polymerase as described herein. Incertain embodiments, the kit further includes one or more additionalcontainers providing one or more additional reagents. For example, inspecific variations, the one or more additional containers provide freenucleotides; a buffer suitable for primer extension; and/or a primerhybridizable, under primer extension conditions, to a predeterminedpolynucleotide template.

Further provided are reaction mixtures comprising the polymerases of theinvention. The reactions mixtures can also contain a template nucleicacid (DNA and/or RNA), one or more primer or probe polynucleotides, freenucleotides (including, e.g., deoxyribonucleotides, ribonucleotides,labeled nucleotides, unconventional nucleotides), buffers, salts, labels(e.g., fluorophores).

Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention pertains. Although essentially anymethods and materials similar to those described herein can be used inthe practice or testing of the present invention, only exemplary methodsand materials are described. For purposes of the present invention, thefollowing terms are defined below.

The terms “a,” “an,” and “the” include plural referents, unless thecontext clearly indicates otherwise.

An “amino acid” refers to any monomer unit that can be incorporated intoa peptide, polypeptide, or protein. As used herein, the term “aminoacid” includes the following twenty natural or genetically encodedalpha-amino acids: alanine (Ala or A), arginine (Arg or R), asparagine(Asn or N), aspartic acid (Asp or D), cysteine (Cys or C), glutamine(Gln or Q), glutamic acid (Glu or E), glycine (Gly or G), histidine (Hisor H), isoleucine (Ile or I), leucine (Leu or L), lysine (Lys or K),methionine (Met or M), phenylalanine (Phe or F), proline (Pro or P),serine (Ser or S), threonine (Thr or T), tryptophan (Trp or W), tyrosine(Tyr or Y), and valine (Val or V). The structures of these twentynatural amino acids are shown in, e.g., Stryer et al., Biochemistry,5^(th) ed., Freeman and Company (2002), which is incorporated byreference. Additional amino acids, such as selenocysteine andpyrrolysine, can also be genetically coded for (Stadtman (1996)“Selenocysteine,” Annu Rev Biochem. 65:83-100 and Ibba et al. (2002)“Genetic code: introducing pyrrolysine,” Curr Biol. 12(13):R464-R466,which are both incorporated by reference). The term “amino acid” alsoincludes unnatural amino acids, modified amino acids (e.g., havingmodified side chains and/or backbones), and amino acid analogs. See,e.g., Zhang et al. (2004) “Selective incorporation of5-hydroxytryptophan into proteins in mammalian cells,” Proc. Natl. Acad.Sci. U.S.A. 101(24):8882-8887, Anderson et al. (2004) “An expandedgenetic code with a functional quadruplet codon” Proc. Natl. Acad. Sci.U.S.A. 101(20):7566-7571, Ikeda et al. (2003) “Synthesis of a novelhistidine analogue and its efficient incorporation into a protein invivo,” Protein Eng. Des. Sel. 16(9):699-706, Chin et al. (2003) “AnExpanded Eukaryotic Genetic Code,” Science 301(5635):964-967, James etal. (2001) “Kinetic characterization of ribonuclease S mutantscontaining photoisomerizable phenylazophenylalanine residues,” ProteinEng. Des. Sel. 14(12):983-991, Kohrer et al. (2001) “Import of amber andochre suppressor tRNAs into mammalian cells: A general approach tosite-specific insertion of amino acid analogues into proteins,” Proc.Natl. Acad. Sci. U.S.A. 98(25):14310-14315, Bacher et al. (2001)“Selection and Characterization of Escherichia coli Variants Capable ofGrowth on an Otherwise Toxic Tryptophan Analogue,” J. Bacteriol.183(18):5414-5425, Hamano-Takaku et al. (2000) “A Mutant Escherichiacoli Tyrosyl-tRNA Synthetase Utilizes the Unnatural Amino AcidAzatyrosine More Efficiently than Tyrosine,” J. Biol. Chem.275(51):40324-40328, and Budisa et al. (2001) “Proteins with{beta}-(thienopyrrolyl)alanines as alternative chromophores andpharmaceutically active amino acids,” Protein Sci. 10(7):1281-1292,which are each incorporated by reference.

To further illustrate, an amino acid is typically an organic acid thatincludes a substituted or unsubstituted amino group, a substituted orunsubstituted carboxy group, and one or more side chains or groups, oranalogs of any of these groups. Exemplary side chains include, e.g.,thiol, seleno, sulfonyl, alkyl, aryl, acyl, keto, azido, hydroxyl,hydrazine, cyano, halo, hydrazide, alkenyl, alkynl, ether, borate,boronate, phospho, phosphono, phosphine, heterocyclic, enone, imine,aldehyde, ester, thioacid, hydroxylamine, or any combination of thesegroups. Other representative amino acids include, but are not limitedto, amino acids comprising photoactivatable cross-linkers, metal bindingamino acids, spin-labeled amino acids, fluorescent amino acids,metal-containing amino acids, amino acids with novel functional groups,amino acids that covalently or noncovalently interact with othermolecules, photocaged and/or photoisomerizable amino acids, radioactiveamino acids, amino acids comprising biotin or a biotin analog,glycosylated amino acids, other carbohydrate modified amino acids, aminoacids comprising polyethylene glycol or polyether, heavy atomsubstituted amino acids, chemically cleavable and/or photocleavableamino acids, carbon-linked sugar-containing amino acids, redox-activeamino acids, amino thioacid containing amino acids, and amino acidscomprising one or more toxic moieties.

The term “mutant,” in the context of DNA polymerases of the presentinvention, means a polypeptide, typically recombinant, that comprisesone or more amino acid substitutions relative to a corresponding,functional DNA polymerase.

The term “unmodified form,” in the context of a mutant polymerase, is aterm used herein for purposes of defining a mutant DNA polymerase of thepresent invention: the term “unmodified form” refers to a functional DNApolymerase that has the amino acid sequence of the mutant polymeraseexcept at one or more amino acid position(s) specified as characterizingthe mutant polymerase. Thus, reference to a mutant DNA polymerase interms of (a) its unmodified form and (b) one or more specified aminoacid substitutions means that, with the exception of the specified aminoacid substitution(s), the mutant polymerase otherwise has an amino acidsequence identical to the unmodified form in the specified motif Thepolymerase may contain additional mutations to provide desiredfunctionality, e.g., improved incorporation of dideoxyribonucleotides,ribonucleotides, ribonucleotide analogs, dye-labeled nucleotides,modulating 5′-nuclease activity, modulating 3′-nuclease (orproofreading) activity, or the like. Accordingly, in carrying out thepresent invention as described herein, the unmodified form of a DNApolymerase is predetermined. The unmodified form of a DNA polymerase canbe, for example, a wild-type and/or a naturally occurring DNApolymerase, or a DNA polymerase that has already been intentionallymodified. An unmodified form of the polymerase is preferably athermostable DNA polymerases, such as DNA polymerases from variousthermophilic bacteria, as well as functional variants thereof havingsubstantial sequence identity to a wild-type or naturally occurringthermostable polymerase Such variants can include, for example, chimericDNA polymerases such as, for example, the chimeric DNA polymerasesdescribed in U.S. Pat. No. 6,228,628 and U.S. Application PublicationNo. 2004/0005599, which are incorporated by reference herein in theirentirety. In certain embodiments, the unmodified form of a polymerasehas reverse transcriptase (RT) activity.

The term “thermostable polymerase,” refers to an enzyme that is stableto heat, is heat resistant, and retains sufficient activity to effectsubsequent primer extension reactions and does not become irreversiblydenatured (inactivated) when subjected to the elevated temperatures forthe time necessary to effect denaturation of double-stranded nucleicacids. The heating conditions necessary for nucleic acid denaturationare well known in the art and are exemplified in, e.g., U.S. Pat. Nos.4,683,202, 4,683,195, and 4,965,188, which are incorporated herein byreference. As used herein, a thermostable polymerase is suitable for usein a temperature cycling reaction such as the polymerase chain reaction(“PCR”). Irreversible denaturation for purposes herein refers topermanent and complete loss of enzymatic activity. For a thermostablepolymerase, enzymatic activity refers to the catalysis of thecombination of the nucleotides in the proper manner to form primerextension products that are complementary to a template nucleic acidstrand. Thermostable DNA polymerases from thermophilic bacteria include,e.g., DNA polymerases from Thermotoga maritima, Thermus aquaticus,Thermus thermophilus, Thermus flavus, Thermus filiformis, Thermusspecies sps17, Thermus species Z05, Thermus caldophilus, Bacilluscaldotenax, Thermotoga neopolitana, and Thermosipho africanus.

As used herein, a “chimeric” protein refers to a protein whose aminoacid sequence represents a fusion product of subsequences of the aminoacid sequences from at least two distinct proteins. A chimeric proteintypically is not produced by direct manipulation of amino acidsequences, but, rather, is expressed from a “chimeric” gene that encodesthe chimeric amino acid sequence. In certain embodiments, for example,an unmodified form of a mutant DNA polymerase of the present inventionis a chimeric protein that consists of an amino-terminal (N-terminal)region derived from a Thermus species DNA polymerase and acarboxy-terminal (C-terminal) region derived from Tma DNA polymerase.The N-terminal region refers to a region extending from the N-terminus(amino acid position 1) to an internal amino acid. Similarly, theC-terminal region refers to a region extending from an internal aminoacid to the C-terminus.

In the context of mutant DNA polymerases, “correspondence” to anothersequence (e.g., regions, fragments, nucleotide or amino acid positions,or the like) is based on the convention of numbering according tonucleotide or amino acid position number and then aligning the sequencesin a manner that maximizes the percentage of sequence identity. Becausenot all positions within a given “corresponding region” need beidentical, non-matching positions within a corresponding region may beregarded as “corresponding positions.” Accordingly, as used herein,referral to an “amino acid position corresponding to amino acid position[X]” of a specified DNA polymerase represents referral to a collectionof equivalent positions in other recognized DNA polymerases andstructural homologues and families. In typical embodiments of thepresent invention, “correspondence” of amino acid positions aredetermined with respect to a region of the polymerase comprising one ormore motifs of SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, as discussedfurther herein.

“Recombinant,” as used herein, refers to an amino acid sequence or anucleotide sequence that has been intentionally modified by recombinantmethods. By the term “recombinant nucleic acid” herein is meant anucleic acid, originally formed in vitro, in general, by themanipulation of a nucleic acid by endonucleases, in a form not normallyfound in nature. Thus an isolated, mutant DNA polymerase nucleic acid,in a linear form, or an expression vector formed in vitro by ligatingDNA molecules that are not normally joined, are both consideredrecombinant for the purposes of this invention. It is understood thatonce a recombinant nucleic acid is made and reintroduced into a hostcell, it will replicate non-recombinantly, i.e., using the in vivocellular machinery of the host cell rather than in vitro manipulations;however, such nucleic acids, once produced recombinantly, althoughsubsequently replicated non-recombinantly, are still consideredrecombinant for the purposes of the invention. A “recombinant protein”is a protein made using recombinant techniques, i.e., through theexpression of a recombinant nucleic acid as depicted above. Arecombinant protein is typically distinguished from naturally occurringprotein by at least one or more characteristics.

A nucleic acid is “operably linked” when it is placed into a functionalrelationship with another nucleic acid sequence. For example, a promoteror enhancer is operably linked to a coding sequence if it affects thetranscription of the sequence; or a ribosome binding site is operablylinked to a coding sequence if it is positioned so as to facilitatetranslation.

The term “host cell” refers to both single-cellular prokaryote andeukaryote organisms (e.g., bacteria, yeast, and actinomycetes) andsingle cells from higher order plants or animals when being grown incell culture.

The term “vector” refers to a piece of DNA, typically double-stranded,which may have inserted into it a piece of foreign DNA. The vector ormay be, for example, of plasmid origin. Vectors contain “replicon”polynucleotide sequences that facilitate the autonomous replication ofthe vector in a host cell. Foreign DNA is defined as heterologous DNA,which is DNA not naturally found in the host cell, which, for example,replicates the vector molecule, encodes a selectable or screenablemarker, or encodes a transgene. The vector is used to transport theforeign or heterologous DNA into a suitable host cell. Once in the hostcell, the vector can replicate independently of or coincidental with thehost chromosomal DNA, and several copies of the vector and its insertedDNA can be generated. In addition, the vector can also contain thenecessary elements that permit transcription of the inserted DNA into anmRNA molecule or otherwise cause replication of the inserted DNA intomultiple copies of RNA. Some expression vectors additionally containsequence elements adjacent to the inserted DNA that increase thehalf-life of the expressed mRNA and/or allow translation of the mRNAinto a protein molecule. Many molecules of mRNA and polypeptide encodedby the inserted DNA can thus be rapidly synthesized.

The term “nucleotide,” in addition to referring to the naturallyoccurring ribonucleotide or deoxyribonucleotide monomers, shall hereinbe understood to refer to related structural variants thereof, includingderivatives and analogs, that are functionally equivalent with respectto the particular context in which the nucleotide is being used (e.g.,hybridization to a complementary base), unless the context clearlyindicates otherwise.

The term “nucleic acid” or “polynucleotide” refers to a polymer that canbe corresponded to a ribose nucleic acid (RNA) or deoxyribose nucleicacid (DNA) polymer, or an analog thereof. This includes polymers ofnucleotides such as RNA and DNA, as well as synthetic forms, modified(e.g., chemically or biochemically modified) forms thereof, and mixedpolymers (e.g., including both RNA and DNA subunits). Exemplarymodifications include methylation, substitution of one or more of thenaturally occurring nucleotides with an analog, internucleotidemodifications such as uncharged linkages (e.g., methyl phosphonates,phosphotriesters, phosphoamidates, carbamates, and the like), pendentmoieties (e.g., polypeptides), intercalators (e.g., acridine, psoralen,and the like), chelators, alkylators, and modified linkages (e.g., alphaanomeric nucleic acids and the like). Also included are syntheticmolecules that mimic polynucleotides in their ability to bind to adesignated sequence via hydrogen bonding and other chemicalinteractions. Typically, the nucleotide monomers are linked viaphosphodiester bonds, although synthetic forms of nucleic acids cancomprise other linkages (e.g., peptide nucleic acids as described inNielsen et al. (Science 254:1497-1500, 1991). A nucleic acid can be orcan include, e.g., a chromosome or chromosomal segment, a vector (e.g.,an expression vector), an expression cassette, a naked DNA or RNApolymer, the product of a polymerase chain reaction (PCR), anoligonucleotide, a probe, and a primer. A nucleic acid can be, e.g.,single-stranded, double-stranded, or triple-stranded and is not limitedto any particular length. Unless otherwise indicated, a particularnucleic acid sequence optionally comprises or encodes complementarysequences, in addition to any sequence explicitly indicated.

The term “oligonucleotide” refers to a nucleic acid that includes atleast two nucleic acid monomer units (e.g., nucleotides). Anoligonucleotide typically includes from about six to about 175 nucleicacid monomer units, more typically from about eight to about 100 nucleicacid monomer units, and still more typically from about 10 to about 50nucleic acid monomer units (e.g., about 15, about 20, about 25, about30, about 35, or more nucleic acid monomer units). The exact size of anoligonucleotide will depend on many factors, including the ultimatefunction or use of the oligonucleotide. Oligonucleotides are optionallyprepared by any suitable method, including, but not limited to,isolation of an existing or natural sequence, DNA replication oramplification, reverse transcription, cloning and restriction digestionof appropriate sequences, or direct chemical synthesis by a method suchas the phosphotriester method of Narang et al. (Meth. Enzymol. 68:90-99,1979); the phosphodiester method of Brown et al. (Meth. Enzymol.68:109-151, 1979); the diethylphosphoramidite method of Beaucage et al.(Tetrahedron Lett. 22:1859-1862, 1981); the triester method of Matteucciet al. (J. Am. Chem. Soc. 103:3185-3191, 1981); automated synthesismethods; or the solid support method of U.S. Pat. No. 4,458,066,entitled “PROCESS FOR PREPARING POLYNUCLEOTIDES,” issued Jul. 3, 1984 toCaruthers et al., or other methods known to those skilled in the art.All of these references are incorporated by reference.

The term “primer” as used herein refers to a polynucleotide capable ofacting as a point of initiation of template-directed nucleic acidsynthesis when placed under conditions in which primer extension isinitiated (e.g., under conditions comprising the presence of requisitenucleoside triphosphates (as dictated by the template that is copied)and a polymerase in an appropriate buffer and at a suitable temperatureor cycle(s) of temperatures (e.g., as in a polymerase chain reaction)).To further illustrate, primers can also be used in a variety of otheroligonucleotide-mediated synthesis processes, including as initiators ofde novo RNA synthesis and in vitro transcription-related processes(e.g., nucleic acid sequence-based amplification (NASBA), transcriptionmediated amplification (TMA), etc.). A primer is typically asingle-stranded oligonucleotide (e.g., oligodeoxyribonucleotide). Theappropriate length of a primer depends on the intended use of the primerbut typically ranges from 6 to 40 nucleotides, more typically from 15 to35 nucleotides. Short primer molecules generally require coolertemperatures to form sufficiently stable hybrid complexes with thetemplate. A primer need not reflect the exact sequence of the templatebut must be sufficiently complementary to hybridize with a template forprimer elongation to occur. In certain embodiments, the term “primerpair” means a set of primers including a 5′ sense primer (sometimescalled “forward”) that hybridizes with the complement of the 5′ end ofthe nucleic acid sequence to be amplified and a 3′ antisense primer(sometimes called “reverse”) that hybridizes with the 3′ end of thesequence to be amplified (e.g., if the target sequence is expressed asRNA or is an RNA). A primer can be labeled, if desired, by incorporatinga label detectable by spectroscopic, photochemical, biochemical,immunochemical, or chemical means. For example, useful labels include³²P, fluorescent dyes, electron-dense reagents, enzymes (as commonlyused in ELISA assays), biotin, or haptens and proteins for whichantisera or monoclonal antibodies are available.

The term “conventional” or “natural” when referring to nucleic acidbases, nucleoside triphosphates, or nucleotides refers to those whichoccur naturally in the polynucleotide being described (i.e., for DNAthese are dATP, dGTP, dCTP and dTTP). Additionally, dITP, and7-deaza-dGTP are frequently utilized in place of dGTP and 7-deaza-dATPcan be utilized in place of dATP in in vitro DNA synthesis reactions,such as sequencing. Collectively, these may be referred to as dNTPs.

The term “unconventional” or “modified” when referring to a nucleic acidbase, nucleoside, or nucleotide includes modification, derivations, oranalogues of conventional bases, nucleosides, or nucleotides thatnaturally occur in a particular polynucleotide. Certain unconventionalnucleotides are modified at the 2′ position of the ribose sugar incomparison to conventional dNTPs. Thus, although for RNA the naturallyoccurring nucleotides are ribonucleotides (i.e., ATP, GTP, CTP, UTP,collectively rNTPs), because these nucleotides have a hydroxyl group atthe 2′ position of the sugar, which, by comparison is absent in dNTPs,as used herein, ribonucleotides are unconventional nucleotides assubstrates for DNA polymerases. As used herein, unconventionalnucleotides include, but are not limited to, compounds used asterminators for nucleic acid sequencing. Exemplary terminator compoundsinclude but are not limited to those compounds that have a 2′,3′ dideoxystructure and are referred to as dideoxynucleoside triphosphates. Thedideoxynucleoside triphosphates ddATP, ddTTP, ddCTP and ddGTP arereferred to collectively as ddNTPs. Additional examples of terminatorcompounds include 2′-PO₄ analogs of ribonucleotides (see, e.g., U.S.Application Publication Nos. 2005/0037991 and 2005/0037398, which areboth incorporated by reference). Other unconventional nucleotidesinclude phosphorothioate dNTPs ([[α]-S]dNTPs), 5′-[α]-borano-dNTPs,[α]-methyl-phosphonate dNTPs, and ribonucleoside triphosphates (rNTPs).Unconventional bases may be labeled with radioactive isotopes such as³²P, ³³P, or ³⁵S; fluorescent labels; chemiluminescent labels;bioluminescent labels; hapten labels such as biotin; or enzyme labelssuch as streptavidin or avidin. Fluorescent labels may include dyes thatare negatively charged, such as dyes of the fluorescein family, or dyesthat are neutral in charge, such as dyes of the rhodamine family, ordyes that are positively charged, such as dyes of the cyanine family.Dyes of the fluorescein family include, e.g., FAM, HEX, TET, JOE, NANand ZOE. Dyes of the rhodamine family include Texas Red, ROX, R110, R6G,and TAMRA. Various dyes or nucleotides labeled with FAM, HEX, TET, JOE,NAN, ZOE, ROX, R110, R6G, Texas Red and TAMRA are marketed byPerkin-Elmer (Boston, Mass.), Applied Biosystems (Foster City, Calif.),or Invitrogen/Molecular Probes (Eugene, Oreg.). Dyes of the cyaninefamily include Cy2, Cy3, Cy5, and Cy7 and are marketed by GE HealthcareUK Limited (Amersham Place, Little Chalfont, Buckinghamshire, England).

As used herein, “percentage of sequence identity” is determined bycomparing two optimally aligned sequences over a comparison window,wherein the portion of the sequence in the comparison window cancomprise additions or deletions (i.e., gaps) as compared to thereference sequence (which does not comprise additions or deletions) foroptimal alignment of the two sequences. The percentage is calculated bydetermining the number of positions at which the identical nucleic acidbase or amino acid residue occurs in both sequences to yield the numberof matched positions, dividing the number of matched positions by thetotal number of positions in the window of comparison and multiplyingthe result by 100 to yield the percentage of sequence identity.

The terms “identical” or percent “identity,” in the context of two ormore nucleic acids or polypeptide sequences, refer to two or moresequences or subsequences that are the same or have a specifiedpercentage of nucleotides or amino acid residues that are the same(e.g., 60% identity, optionally 65%, 70%, 75%, 80%, 85%, 90%, or 95%identity over a specified region), when compared and aligned for maximumcorrespondence over a comparison window, or designated region asmeasured using one of the following sequence comparison algorithms or bymanual alignment and visual inspection. Sequences are “substantiallyidentical” to each other if they are at least 20%, at least 25%, atleast 30%, at least 35%, at least 40%, at least 45%, at least 50%, or atleast 55% identical. These definitions also refer to the complement of atest sequence. Optionally, the identity exists over a region that is atleast about 50 nucleotides in length, or more typically over a regionthat is 100 to 500 or 1000 or more nucleotides in length.

The terms “similarity” or “percent similarity,” in the context of two ormore polypeptide sequences, refer to two or more sequences orsubsequences that have a specified percentage of amino acid residuesthat are either the same or similar as defined by a conservative aminoacid substitutions (e.g., 60% similarity, optionally 65%, 70%, 75%, 80%,85%, 90%, or 95% similar over a specified region), when compared andaligned for maximum correspondence over a comparison window, ordesignated region as measured using one of the following sequencecomparison algorithms or by manual alignment and visual inspection.Sequences are “substantially similar” to each other if they are at least20%, at least 25%, at least 30%, at least 35%, at least 40%, at least45%, at least 50%, or at least 55% similar to each other. Optionally,this similarly exists over a region that is at least about 50 aminoacids in length, or more typically over a region that is at least about100 to 500 or 1000 or more amino acids in length.

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are entered into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. Default programparameters are commonly used, or alternative parameters can bedesignated. The sequence comparison algorithm then calculates thepercent sequence identities or similarities for the test sequencesrelative to the reference sequence, based on the program parameters.

A “comparison window,” as used herein, includes reference to a segmentof any one of the number of contiguous positions selected from the groupconsisting of from 20 to 600, usually about 50 to about 200, moreusually about 100 to about 150 in which a sequence may be compared to areference sequence of the same number of contiguous positions after thetwo sequences are optimally aligned. Methods of alignment of sequencesfor comparison are well known in the art. Optimal alignment of sequencesfor comparison can be conducted, for example, by the local homologyalgorithm of Smith and Waterman (Adv. Appl. Math. 2:482, 1970), by thehomology alignment algorithm of Needleman and Wunsch (J. Mol. Biol.48:443, 1970), by the search for similarity method of Pearson and Lipman(Proc. Natl. Acad. Sci. USA 85:2444, 1988), by computerizedimplementations of these algorithms (e.g., GAP, BESTFIT, FASTA, andTFASTA in the Wisconsin Genetics Software Package, Genetics ComputerGroup, 575 Science Dr., Madison, Wis.), or by manual alignment andvisual inspection (see, e.g., Ausubel et al., Current Protocols inMolecular Biology (1995 supplement)).

One example of a useful algorithm is PILEUP. PILEUP creates a multiplesequence alignment from a group of related sequences using progressive,pairwise alignments to show relationship and percent sequence identity.It also plots a tree or dendogram showing the clustering relationshipsused to create the alignment. PILEUP uses a simplification of theprogressive alignment method of Feng and Doolittle (J. Mol. Evol.35:351-360, 1987). The method used is similar to the method described byHiggins and Sharp (CABIOS 5:151-153, 1989). The program can align up to300 sequences, each of a maximum length of 5,000 nucleotides or aminoacids. The multiple alignment procedure begins with the pairwisealignment of the two most similar sequences, producing a cluster of twoaligned sequences. This cluster is then aligned to the next most relatedsequence or cluster of aligned sequences. Two clusters of sequences arealigned by a simple extension of the pairwise alignment of twoindividual sequences. The final alignment is achieved by a series ofprogressive, pairwise alignments. The program is run by designatingspecific sequences and their amino acid or nucleotide coordinates forregions of sequence comparison and by designating the programparameters. Using PILEUP, a reference sequence is compared to other testsequences to determine the percent sequence identity relationship usingthe following parameters: default gap weight (3.00), default gap lengthweight (0.10), and weighted end gaps. PILEUP can be obtained from theGCG sequence analysis software package (e.g., version 7.0 (Devereaux etal., Nuc. Acids Res. 12:387-95, 1984) or later).

Another example of an algorithm that is suitable for determining percentsequence identity and sequence similarity are the BLAST and BLAST 2.0algorithms, which are described in Altschul et al. (Nuc. Acids Res.25:3389-402, 1977), and Altschul et al. (J. Mol. Biol. 215:403-10,1990), respectively. Software for performing BLAST analyses is publiclyavailable through the National Center for Biotechnology Information(http://www.ncbi.nlm.nih.gov/). This algorithm involves firstidentifying high scoring sequence pairs (HSPs) by identifying shortwords of length W in the query sequence, which either match or satisfysome positive-valued threshold score T when aligned with a word of thesame length in a database sequence. T is referred to as the neighborhoodword score threshold (Altschul et al., supra). These initialneighborhood word hits act as seeds for initiating searches to findlonger HSPs containing them. The word hits are extended in bothdirections along each sequence for as far as the cumulative alignmentscore can be increased. Cumulative scores are calculated using, fornucleotide sequences, the parameters M (reward score for a pair ofmatching residues; always >0) and N (penalty score for mismatchingresidues; always <0). For amino acid sequences, a scoring matrix is usedto calculate the cumulative score. Extension of the word hits in eachdirection are halted when: the cumulative alignment score falls off bythe quantity X from its maximum achieved value; the cumulative scoregoes to zero or below, due to the accumulation of one or morenegative-scoring residue alignments; or the end of either sequence isreached. The BLAST algorithm parameters W, T, and X determine thesensitivity and speed of the alignment. The BLASTN program (fornucleotide sequences) uses as defaults a wordlength (W) of 11, anexpectation (E) or 10, M=5, N=−4 and a comparison of both strands. Foramino acid sequences, the BLASTP program uses as defaults a wordlengthof 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (seeHenikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915, 1989)alignments (B) of 50, expectation (E) of 10, M=5, N=−4, and a comparisonof both strands.

The BLAST algorithm also performs a statistical analysis of thesimilarity between two sequences (see, e.g., Karlin and Altschul, Proc.Natl. Acad. Sci. USA 90:5873-87, 1993). One measure of similarityprovided by the BLAST algorithm is the smallest sum probability (P(N)),which provides an indication of the probability by which a match betweentwo nucleotide or amino acid sequences would occur by chance. Forexample, a nucleic acid is considered similar to a reference sequence ifthe smallest sum probability in a comparison of the test nucleic acid tothe reference nucleic acid is less than about 0.2, typically less thanabout 0.01, and more typically less than about 0.001.

The term “nucleic acid extension rate” refers the rate at which abiocatalyst (e.g., an enzyme, such as a polymerase, ligase, or the like)extends a nucleic acid (e.g., a primer or other oligonucleotide) in atemplate-dependent or template-independent manner by attaching (e.g.,covalently) one or more nucleotides to the nucleic acid. To illustrate,certain mutant DNA polymerases described herein have improved nucleicacid extension rates relative to unmodified forms of these DNApolymerases, such that they can extend primers at higher rates thanthese unmodified forms under a given set of reaction conditions.

The term “reverse transcription efficiency” refers to the fraction ofRNA molecules that are reverse transcribed as cDNA in a given reversetranscription reaction. In certain embodiments, the mutant DNApolymerases of the invention have improved reverse transcriptionefficiencies relative to unmodified forms of these DNA polymerases. Thatis, these mutant DNA polymerases reverse transcribe a higher fraction ofRNA templates than their unmodified forms under a particular set ofreaction conditions.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts an amino acid sequence alignment of a region from thepolymerase domain of exemplary thermostable DNA polymerases from variousspecies of thermophilic bacteria: Thermus thermophilus (Tth) (SEQ IDNO:4), Thermus caldophilus (Tca) (SEQ ID NO:5), Thermus species Z05(Z05) (SEQ ID NO:6), Thermus aquaticus (Taq) (SEQ ID NO:7), Thermusflavus (Tfl) (SEQ ID NO:8), Thermus filiformis (Tfi) (SEQ ID NO:9),Thermus species sps17 (Sps17) (SEQ ID NO:10), Thermotoga maritima (Tma)(SEQ ID NO:11), Thermotoga neapolitana (Tne) (SEQ ID NO:12), Thermosiphoafricanus (Taf) (SEQ ID NO:13), and Bacillus caldotenax (Bca) (SEQ IDNO:14). The amino acid sequence alignment also includes a region fromthe polymerase domain of representative chimeric thermostable DNApolymerases, namely, CS5 (SEQ ID NO:15) and CS6 (SEQ ID NO:16). Inaddition, a sequence (Cons) (SEQ ID NO:17) showing consensus amino acidresidues among these exemplary sequences is also included. Further, thepolypeptide regions shown comprise the amino acid motifsXXXXRXXXKLXXTYXX (SEQ ID NO:1), TGRLSSXXPNLQN (SEQ ID NO:2), andXXXXXXXDYSQIELR (SEQ ID NO:3), the variable positions of which arefurther defined herein. These motifs are highlighted in bold type foreach polymerase sequence. Amino acid positions amenable to mutation inaccordance with the present invention are indicated with an asterisk(*). Gaps in the alignments are indicated with a dot (.).

FIG. 2A presents the amino acid sequence of the chimeric thermostableDNA polymerase CS5 (SEQ ID NO:18).

FIG. 2B presents a nucleic acid sequence encoding the chimericthermostable DNA polymerase CS5 (SEQ ID NO:20).

FIG. 3A presents the amino acid sequence of the chimeric thermostableDNA polymerase CS6 (SEQ ID NO:19).

FIG. 3B presents a nucleic acid sequence encoding the chimericthermostable DNA polymerase CS6 (SEQ ID NO:21).

FIG. 4 is a bar graph that shows the normalized extension rates ofvarious mutants of a G46E L329A E678G (GLE) CS5 DNA polymerase. They-axis represents the relative extension rates, while the x-axisrepresents the DNA polymerases having specified point mutations (G=G46E,L=L329A, Q=Q601R, D=D640G, I=I669F, S=S671F, and E=E678G). The extensionrate values obtained for the mutant polymerases are normalized relativeto the value obtained for the GLE CS5 DNA polymerase, which is set to1.00.

FIG. 5 is a bar graph that shows the normalized extension rates ofvarious mutants of a G46E L329A E678G (GLE) CS5 DNA polymerase. They-axis represents the relative extension rates, while the x-axisrepresents the DNA polymerases having specified point mutations (G=G46E,L=L329A, Q=Q601R, D=D640G, I=I669F, S=S671F, and E=E678G). The extensionrate values obtained for the mutant polymerases are normalized relativeto the value obtained for the GLE CS5 DNA polymerase, which is set to1.00.

FIG. 6 is a bar graph that shows the normalized extension rates of a Z05DNA polymerase, a ΔZ05 DNA (dZ05 in FIG. 6) polymerase (see, e.g., U.S.Pat. No. 5,455,170, entitled “MUTATED THERMOSTABLE NUCLEIC ACIDPOLYMERASE ENZYME FROM THERMUS SPECIES Z05” issued Oct. 3, 1995 toAbramson et al. and U.S. Pat. No. 5,674,738, entitled “DNA ENCODINGTHERMOSTABLE NUCLEIC ACID POLYMERASE ENZYME FROM THERMUS SPECIES Z05”issued Oct. 7, 1997 to Abramson et al., which are both incorporated byreference), and various mutants of a G46E L329A (GL) CS5 DNA polymerase.The y-axis represents the relative extension rates, while the x-axisrepresents the DNA polymerases having specified point mutations (G=G46E,L=L329A, Q=Q601R, D=D640G, I=I669F, S=S671F, and E=E678G). The extensionrate values obtained for the mutant polymerases are normalized relativeto the value obtained for a GLE CS5 DNA polymerase, which is set to1.00.

FIG. 7 is a bar graph that shows the normalized extension rates of a Z05DNA polymerase, a ΔZ05 (dZ05 in FIG. 7) DNA polymerase, and variousmutants of a G46E L329A (GL) CS5 DNA polymerase. The y-axis representsthe relative extension rates, while the x-axis represents the DNApolymerases having specified point mutations (G=G46E, L=L329A, Q=Q601R,D=D640G, I=I669F, S=S671F, and E=E678G). The extension rate valuesobtained for the mutant polymerases are normalized relative to the valueobtained for a GLE CS5 DNA polymerase, which is set to 1.00.

FIG. 8 is a plot that shows the extension rates of different DNApolymerases under varied salt (KOAc) concentrations. The y-axisrepresents the extension rates (Arbitrary Units), while the x-axisrepresents KOAc concentration (mM). The legend that accompanies the plotshows the DNA polymerase corresponding to each trace in the plot. Inparticular, delta Z05 refers to A Z05 DNA polymerase and Z05 refers toZ05 DNA polymerase, while the other enzymes indicated refer to mutantCS5 DNA polymerases having specified point mutations (G=G46E, L=L329A,Q=Q601R, D=D640G, S=S671F, and E=E678G).

FIG. 9 is a plot that shows the extension rates of different DNApolymerases under varied salt (KOAc) concentrations. The y-axisrepresents the extension rates (Arbitrary Units), while the x-axisrepresents KOAc concentration (mM). The legend that accompanies the plotshows the DNA polymerase corresponding to each trace in the plot. Inparticular, the other enzymes indicated refer to mutant CS5 DNApolymerases having specified point mutations (G=G46E, L=L329A, Q=Q601R,D=D640G, S=S671F, and E=E678G).

FIG. 10 is a bar graph that shows the threshold cycle (Ct) valuesobtained for various mutant CS5 DNA polymerases in RT-PCRs. The y-axisrepresents the Ct values, while the x-axis represents the DNApolymerases having specified point mutations (G=G46E, L=L329A, Q=Q601R,D=D640G, and S=S671F).

FIG. 11 is a bar graph that shows the threshold cycle (Ct) valuesobtained for various mutant CS5 DNA polymerases in Mg⁺²-activatedRT-PCRs having varied RT incubation times. The y-axis represents the Ctvalues, while the x-axis represents the DNA polymerases having specifiedpoint mutations (G=G46E, L=L329A, Q=Q601R, D=D640G, and S=S671F).

FIG. 12 is a bar graph that shows the Ct values obtained for variousmutant CS5 DNA polymerases in Mn⁺²-activated RT-PCRs having varied RTincubation times. The y-axis represents the Ct values, while the x-axisrepresents the DNA polymerases having specified point mutations (G=G46E,L=L329A, Q=Q601R, D=D640G, and S=S671F).

FIGS. 13A and 13B are photographs of agarose gels that illustrate theability of certain enzymes described herein to make full length ampliconunder the various conditions involving ribonucleotides. As labeled onthe photographs, the enzymes tested were GQDSE, CS6-GQDSE, GLQDSE, GDSE,GLDSE, GLDE, GE (G46E CSSR), and a 4:1 mixture of GL and GLE (GLCS5/GLE), where G=G46E, L=L329A, Q=Q601R, D=D640G, S=S671F, and E=E678G.All of the enzymes were CS5 enzymes aside from the one denotedCS6-GQDSE.

FIG. 14A is a plot of delta Cts (y-axis) for the enzymes described withrespect to FIGS. 13A and 13B against various rATP conditions tested(y-axis), while FIG. 14B is a plot of % rNTP incorporation (y-axis) forthe enzymes described with respect to FIGS. 13A and 13B against variousrNTP conditions tested (y-axis).

FIGS. 15A and 15B are photographs of agarose gels that illustrate theability of certain enzymes described herein to make full length ampliconunder the various conditions involving biotinylated ribonucleotides. Aslabeled on the photographs, the CS5 enzymes tested were GQDSE, GDSE, GE(G46E CSSR), and a 4:1 mixture of GL and GLE (GL/GLE Blend (4:1)), whereG=G46E, L=L329A, Q=Q601R, D=D640G, S=S671F, and E=E678G.

FIG. 16A is a plot of delta Cts (y-axis) for the enzymes (x-axis)described with respect to FIGS. 15A and 15B for various rCTP conditionstested (legend), while FIG. 16B is a plot of delta Cts (y-axis) forthose enzymes (x-axis) for various biotin labeled rCTP conditions tested(legend).

FIG. 17 is a bar graph that shows the effect of enzyme concentration onthreshold cycle (Ct) values in pyrophosphorolysis activatedpolymerization (PAP) reactions utilizing a G46E L329A E678G (GLE) CS5DNA polymerase. The y-axis represents Ct value, while the x-axisrepresents the enzyme concentration (nM). The legend that accompaniesthe plot shows the number of copies of the template nucleic acidcorresponding to each trace in the graph (no copies of the templatenucleic acid (no temp), 1e⁴ copies of the template nucleic acid(1E4/r×n), 1e⁵ copies of the template nucleic acid (1E5/r×n), and 1e⁶copies of the template nucleic acid (1E6/r×n)).

FIG. 18 is a bar graph that shows the effect of enzyme concentration onthreshold cycle (Ct) values in pyrophosphorolysis activatedpolymerization (PAP) reactions utilizing a G46E L329A D640G S671F E678G(GLDSE) CS5 DNA polymerase. The y-axis represents Ct value, while thex-axis represents the enzyme concentration (nM). The legend thataccompanies the plot shows the number of copies of the template nucleicacid corresponding to each trace in the graph (no copies of the templatenucleic acid (no temp), 1e⁴ copies of the template nucleic acid(1E4/r×n), 1e⁵ copies of the template nucleic acid (1E5/r×n), and 1e⁶copies of the template nucleic acid (1E6/r×n)).

FIG. 19 is a bar graph that shows the normalized extension rates ofvarious mutants of a Thermus sp. Z05 DNA polymerase. The y-axisrepresents the relative extension rates, while the x-axis representsThermus sp. Z05 DNA polymerase (Z05) and various Z05 DNA polymeraseshaving specified point mutations (Q=T541R, D=D580G, and S=A610F). Thex-axis represents also represents ES112 (E683R Z05 DNA polymerase; see,U.S. Pat. Appl. No. 20020012970, entitled “High temperature reversetranscription using mutant DNA polymerases” filed Mar. 30, 2001 by Smithet al., which is incorporated by reference) and ES112-D (D580G E683R Z05DNA polymerase). The extension rate values obtained for the mutantpolymerases are normalized relative to the value obtained for the Z05DNA polymerase, which is set to 1.00.

FIG. 20 is a photograph of a gel that shows the detection of PCRproducts from an analysis that involved PAP-related HIV DNA templatetitrations.

FIG. 21 is a graph that shows threshold cycle (C_(T)) values observedfor various mutant K-Ras plasmid template copy numbers utilized inamplifications that involved blocked or unblocked primers.

FIG. 22 is a graph that shows threshold cycle (C_(T)) values observedfor various enzymes and enzyme concentrations utilized in amplificationsthat involved a K-Ras plasmid template.

FIG. 23 is a bar graph that shows data for PAP reverse transcriptionreactions on HCV RNA in which products of the cDNA reaction weremeasured using a quantitative PCR assay specific for the HCV cDNA. They-axis represents Ct value, while the x-axis represents the Units ofenzyme utilized in the reactions. As indicated, the enzymes used inthese reactions were Z05 DNA polymerase (Z05) or blends of G46E L329AQ601R D640G S671F E678G (GLQDSE) and G46E L329A Q601R D640G S671F(GLQDS) CS5 DNA polymerases.

FIG. 24 shows PCR growth curves of BRAF oncogene amplifications thatwere generated when bidirectional PAP was performed. The x-axis showsnormalized, accumulated fluorescence and the y-axis shows cycles of PAPPCR amplification.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides novel mutant DNA polymerases in which oneor more amino acids in the polymerase domain have been mutated relativeto a functional DNA polymerase. The mutant DNA polymerases of theinvention are active enzymes having improved rates of nucleotideincorporation relative to the unmodified form of the polymerase and, incertain embodiments, concomitant increases in reverse transcriptaseactivity and/or amplification ability. The mutant DNA polymerases may beused at lower concentrations for superior or equivalent performance asthe parent enzymes. In certain embodiments, the mutant DNA polymerasesdescribed herein have improved thermostability relative to parentenzymes. The mutant DNA polymerases are therefore useful in a variety ofapplications involving primer extension as well as reverse transcriptionor amplification of polynucleotide templates, including, for example,applications in recombinant DNA studies and medical diagnosis ofdisease.

Unmodified forms of DNA polymerases amenable to mutation in accordancewith the present invention are those having a functional polymerasedomain comprising the following amino acid motifs:

-   -   (a)        Xaa-Xaa-Xaa-Xaa-Arg-Xaa-Xaa-Xaa-Lys-Leu-Xaa-Xaa-Thr-Tyr-Xaa-Asp        (also referred to herein in the one-letter code as        X_(a1)-X_(a2)-X_(a3)-X_(a4)-R—X_(a6)-X_(a7)-X_(a8)-K-L-X_(a11)-X_(a12)-T-Y—X_(a15)-X_(a16)        (SEQ ID NO:1)); wherein        -   X_(a1) is Ile (I) or Leu (L);        -   X_(a2) is Gln (Q) or Leu (L);        -   X_(a3) is Gln (Q), His (H) or Glu (E);        -   X_(a4) is Tyr (Y), His (H), or Phe (F);        -   X_(a6) is Glu (E), Gln (Q) or Lys (K);        -   X_(a7) is Ile (I), Leu (L) or Tyr (Y);        -   X_(a8) is Gln (Q), Thr (T), Met (M), Gly (G) or Leu (L);        -   X_(a11) is Lys (K) or Gln (Q);        -   X_(a12) is Ser (S) or Asn (N);        -   X_(a15) is Ile (I) or Val (V); and        -   X_(a16) is Glu (E) or Asp (D);    -   (b) Thr-Gly-Arg-Leu-Ser-Ser-Xaa-Xaa-Pro-Asn-Leu-Gln-Asn (also        referred to herein in the one-letter code as        T-G-R-L-S-S-X_(b7)-X_(b8)-P-N-L-Q-N (SEQ ID NO:2));        -   wherein        -   X_(b7) is Ser (S) or Thr (T);        -   X_(b8) is Asp (D), Glu (E) or Asn (N); and    -   (c) Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Asp-Tyr-Ser-Gln-Ile-Glu-Leu-Arg        (also referred to herein in the one-letter code as        X_(c1)-X_(c2)-X_(c3)-X_(c4)-X_(c5)-X_(c6)-X_(c7)-D-Y-S-Q-I-E-L-R        (SEQ ID NO:3); wherein        -   X_(c1) is Gly (G), Asn (N), or Asp (D);        -   X_(c2) is Trp (W) or His (H);        -   X_(c3) is Trp (W), Ala (A), Leu (L) or Val (V);        -   X_(c4) is Ile (I) or Leu (L);        -   X_(c5) is Val (V), Phe (F) or Leu (L);        -   X_(c6) is Ser (S), Ala (A), Val (V) or Gly (G); and        -   X_(c7) is Ala (A) or Leu (L).            These motifs are present within a region of about 100 amino            acids in the active site of many Family A type DNA-dependent            DNA polymerases, particularly thermostable DNA polymerases            from thermophilic bacteria. For example, FIG. 1 shows an            amino acid sequence alignment of a region from the            polymerase domain of DNA polymerases from several species of            thermophilic bacteria: Thermotoga maritima, Thermus            aquaticus, Thermus thermophilus, Thermus flavus, Thermus            filiformis, Thermus sp. sps17, Thermus sp. Z05, Thermotoga            neopolitana, Thermosipho africanus, Bacillus caldotenax and            Thermus caldophilus. The amino acid sequence alignment shown            in FIG. 1 also includes a region from the polymerase domain            of representative chimeric thermostable DNA polymerases. As            shown, each of the motifs of SEQ ID NOS:1, 2, and 3 is            present in each of these polymerases, indicating a conserved            function for these regions of the active site.

Accordingly, in some embodiments, the unmodified form of the DNApolymerase is a wild-type or a naturally occurring DNA polymerase, suchas, for example, a polymerase from any of the species of thermophilicbacteria listed above. In one variation, the unmodified polymerase isfrom a species of the genus Thermus. In other embodiments of theinvention, the unmodified polymerase is from a thermophilic speciesother than Thermus. The full nucleic acid and amino acid sequence fornumerous thermostable DNA polymerases are available. The sequences eachof Thermus aquaticus (Taq) (SEQ ID NO:78), Thermus thermophilus (Tth)(SEQ ID NO:79), Thermus species Z05 (SEQ ID NO:82), Thermus speciessps17 (SEQ ID NO:81), Thermotoga maritima (Tma) (SEQ ID NO:77), andThermosipho africanus (Taf) (SEQ ID NO:83) polymerase have beenpublished in PCT International Patent Publication No. WO 92/06200, whichis incorporated herein by reference. The sequence for the DNA polymerasefrom Thermus flavus (SEQ ID NO:80) has been published in Akhmetzjanovand Vakhitov (Nucleic Acids Research 20:5839, 1992), which isincorporated herein by reference. The sequence of the thermostable DNApolymerase from Thermus caldophilus (SEQ ID NO:84) is found inEMBL/GenBank Accession No. U62584. The sequence of the thermostable DNApolymerase from Thermus filiformis can be recovered from ATCC DepositNo. 42380 using, e.g., the methods provided in U.S. Pat. No. 4,889,818,as well as the sequence information provided in Table 1. The sequence ofthe Thermotoga neapolitana DNA polymerase (SEQ ID NO:85) is from GeneSeqPatent Data Base Accession No. R98144 and PCT WO 97/09451, eachincorporated herein by reference. The sequence of the thermostable DNApolymerase from Bacillus caldotenax is described in, e.g., Uemori et al.(J Biochem (Tokyo) 113(3):401-410, 1993; see also, Swiss-Prot databaseAccession No. Q04957 and GenBank Accession Nos. D12982 and BAA02361),which are each incorporated by reference. Examples of unmodified formsof DNA polymerases that can be modified as described herein are alsodescribed in, e.g., U.S. Pat. No. 6,228,628, entitled “Mutant chimericDNA polymerase” issued May 8, 2001 to Gelfand et al.; U.S. Pat. No.6,346,379, entitled “Thermostable DNA polymerases incorporatingnucleoside triphosphates labeled with fluorescein family dyes” issuedFeb. 12, 2002 to Gelfand et al.; U.S. Pat. No. 7,030,220, entitled“Thermostable enzyme promoting the fidelity of thermostable DNApolymerases—for improvement of nucleic acid synthesis and amplificationin vitro” issued Apr. 18, 2006 to Ankenbauer et al.; U.S. Pat. No.6,881,559, entitled “Mutant B-type DNA polymerases exhibiting improvedperformance in PCR” issued Apr. 19, 2005 to Sobek et al.; U.S. Pat. No.6,794,177, entitled “Modified DNA-polymerase from carboxydothermushydrogenoformans and its use for coupled reverse transcription andpolymerase chain reaction” issued Sep. 21, 2004 to Markau et al.; U.S.Pat. No. 6,468,775, entitled “Thermostable DNA polymerase fromcarboxydothermus hydrogenoformans” issued Oct. 22, 2002 to Ankenbauer etal.; and U.S. Pat. Appl. Nos. 20040005599, entitled “Thermostable orthermoactive DNA polymerase molecules with attenuated 3′-5′ exonucleaseactivity” filed Mar. 26, 2003 by Schoenbrunner et al.; 20020012970,entitled “High temperature reverse transcription using mutant DNApolymerases” filed Mar. 30, 2001 by Smith et al.; 20060078928, entitled“Thermostable enzyme promoting the fidelity of thermostable DNApolymerases—for improvement of nucleic acid synthesis and amplificationin vitro” filed Sep. 29, 2005 by Ankenbauer et al.; 20040115639,entitled “Reversibly modified thermostable enzymes for DNA synthesis andamplification in vitro” filed Dec. 11, 2002 by Sobek et al., which areeach incorporated by reference.

Also amenable to the mutations described herein are functional DNApolymerases that have been previously modified (e.g., by amino acidsubstitution, addition, or deletion), provided that the previouslymodified polymerase retains the amino acid motifs of SEQ ID NOS:1, 2,and 3. Thus, suitable unmodified DNA polymerases also include functionalvariants of wild-type or naturally occurring polymerases. Such variantstypically will have substantial sequence identity or similarity to thewild-type or naturally occurring polymerase, typically at least 80%sequence identity and more typically at least 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98% or 99% sequence identity. In certain embodiments, theunmodified DNA polymerase has reverse transcriptase (RT) activity and/orthe ability to incorporate ribonucleotides or other 2′-modifiednucleotides.

Suitable polymerases also include, for example, certain chimeric DNApolymerases comprising polypeptide regions from two or more enzymes.Examples of such chimeric DNA polymerases are described in, e.g., U.S.Pat. No. 6,228,628, which is incorporated by reference herein in itsentirety. Particularly suitable are chimeric CS-family DNA polymerases,which include the CS5 (SEQ ID NO:18) and CS6 (SEQ ID NO:19) polymerasesand variants thereof having substantial sequence identity or similarityto SEQ ID NO:18 or SEQ ID NO:19 (typically at least 80% sequenceidentity and more typically at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98% or 99% sequence identity). The CS5 and CS6 DNA polymerases arechimeric enzymes derived from Thermus sp. Z05 and Thermotoga maritima(Tma) DNA polymerases. They comprise the N-terminal 5′-nuclease domainof the Thermus enzyme and the C-terminal 3′-5′ exonuclease and thepolymerase domains of the Tma enzyme. These enzymes have efficientreverse transcriptase activity, can extend nucleotide analog-containingprimers, and can incorporate alpha-phosphorothioate dNTPs, dUTP, dITP,and also fluorescein- and cyanine-dye family labeled dNTPs. The CS5 andCS6 polymerases are also efficient Mg²⁺-activated PCR enzymes. Nucleicacid sequences encoding CS5 and CS6 polymerases are provided in FIGS. 2Band 3B, respectively. CS5 and CS6 chimeric polymerases are furtherdescribed in, e.g., U.S. Pat. Application Publication No. 2004/0005599,which is incorporated by reference herein in its entirety.

In some embodiments, the unmodified form of the DNA polymerase is apolymerase that has been previously modified, typically by recombinantmeans, to confer some selective advantage. Such modifications include,for example, the amino acid substitutions G46E, L329A, and/or E678G inCS5 DNA polymerase, CS6 DNA polymerase, or corresponding mutation(s) inother polymerases. Accordingly, in specific variations, the unmodifiedform of the DNA polymerase is one of the following (each having theamino acid sequence of SEQ ID NO:18 or SEQ ID NO:19 except for thedesignated substitution(s)): G46E; G46E L329A; G46E E678G; or G46E L329AE678G. The E678G substitution, for example, allows for the incorporationof ribonucleotides and other 2′-modified nucleotides, but this mutationalso appears to result in an impaired ability to extend primedtemplates. In certain embodiments, the mutations according to thepresent invention, which result in a faster extension rate of the mutantpolymerase, ameliorate this particular feature of the E678G mutation.

The mutant DNA polymerases of the present invention comprise one or moreamino acid substitutions in the active site relative to the unmodifiedpolymerase. In some embodiments, the amino acid substitution(s) are atat least one of the following amino acid positions:

-   -   position X_(a8) of the motif set forth in SEQ ID NO:1;    -   position X_(b8) of the motif set forth in SEQ ID NO:2;    -   position X_(c4) of the motif set forth in SEQ ID NO:3; and    -   position X_(c6) of the motif set forth in SEQ ID NO:3.        Amino acid substitution at one or more of these positions        confers improved nucleotide-incorporating activity, yielding a        mutant DNA polymerase with an improved (faster) nucleic acid        extension rate relative to the unmodified polymerase. In        addition, amino acid substitution at one or more of these        positions confers increased 3′-5′ exonuclease (proofreading)        activity relative to the unmodified polymerase. While not        intending to be limited to any particular theory, the present        inventors believe that the improved nucleic acid extension rate        of the mutant polymerases of the invention is a consequence of        tighter binding to a template, i.e., less frequent dissociation        from the template, resulting in a higher “processivity” enzyme.        These features permit using lower concentrations of the mutant        polymerase in, e.g., primer extension reactions relative to        reactions involving the unmodified DNA polymerase. Thus, at a        sufficiently high enzyme concentration, the extension rate of        the unmodified polymerase (i.e., lacking the specific mutations        that are the subject of the invention) could conceivably        approach that of the mutant enzyme. The mutant polymerases also        appear to perform much better than the unmodified forms at high        ionic strength. However, at a sufficiently high enzyme        concentration, the performance of the unmodified polymerase at        low ionic strength would approach that of the mutant polymerase.

Because the unmodified forms of DNA polymerase are unique, the aminoacid position corresponding to each of X_(a8), X_(b8), X_(c4), andX_(c6) is typically distinct for each mutant polymerase. Amino acid andnucleic acid sequence alignment programs are readily available (see,e.g., those referred to supra) and, given the particular motifsidentified herein, serve to assist in the identification of the exactamino acids (and corresponding codons) for modification in accordancewith the present invention. The positions corresponding to each ofX_(a8), X_(b8), X_(c4), and X_(c6) are shown in Table 1 forrepresentative chimeric thermostable DNA polymerases and thermostableDNA polymerases from exemplary thermophilic species.

TABLE 1 Amino Acid Positions Corresponding to Motif Positions X_(a8),X_(b8), X_(c4), and X_(c6) in Exemplary Thermostable Polymerases.Organism or Chimeric Sequence Amino Acid Position Consensus X_(a8)X_(b8) X_(c4) X_(c6) T. thermophilus 541 580 608 610 T. caldophilus 541580 608 610 T. sp. Z05 541 580 608 610 T. aquaticus 539 578 606 608 T.flavus 538 577 605 607 T. filiformis 537 576 604 606 T. sp. sps17 537576 604 606 T. maritima 601 640 669 671 T. neapolitana 601 640 669 671T. africanus 600 639 668 670 B. caldotenax 582 621 650 652 CS5 601 640669 671 CS6 601 640 669 671

In some embodiments, the amino acid substitutions are single amino acidsubstitutions. The mutant polymerase can, e.g., comprise any one of theamino acid substitutions at position X_(a8), X_(b8), X_(c4), or X_(c6)separately. Alternatively, the mutant polymerase comprises any one ofvarious combinations of substitutions at two, three, or all four ofthese positions. For example, in one embodiment, the mutant DNApolymerase of the invention comprises amino acid substitutions at eachof positions X_(b8) and X_(c6). Typically, the amino acid at positionX_(a8), X_(b8), X_(c4), or X_(c6) is substituted with an amino acid thatdoes not correspond to the respective motif as set forth in SEQ ID NO:1,SEQ ID NO:2, or SEQ ID NO:3. Thus, typically, the amino acid at positionX_(a8), if substituted, is not Q, T, M, G or L; the amino acid atposition X_(b8), if substituted, is not D, E or N; the amino acid atposition X_(c4), if substituted, is not I or L; and/or the amino acid atposition X_(c6), if substituted, is not S, A, V or G. In certainembodiments, amino acid substitutions include Arginine (R) at positionX_(a8), Glycine (G) at position X_(b8), Phenylalanine (F) at positionX_(c4), and/or Phenylalanine (F) at position X_(c6). Other suitableamino acid substitution(s) at one or more of the identified sites can bedetermined using, e.g., known methods of site-directed mutagenesis anddetermination of primer extension performance in assays describedfurther herein or otherwise known to persons of skill in the art.

As previously discussed, in some embodiments, the mutant DNA polymeraseof the present invention is derived from CS5 DNA polymerase (SEQ IDNO:18), CS6 DNA polymerase (SEQ ID NO:19), or a variant of thosepolymerases (e.g., G46E; G46E L329A; G46E E678G; G46E L329A E678G; orthe like). As referred to above, in CS5 DNA polymerase or CS6 DNApolymerase, position X_(a8) corresponds to Glutamine (Q) at position601; position X_(b8) corresponds to Aspartate (D) at position 640;position X_(c4) corresponds to Isoleucine (I) at position 669; andposition X_(c6) corresponds to Serine (S) at position 671. Thus, incertain variations of the invention, the mutant polymerase comprises atleast one amino acid substitution, relative to a CS5 DNA polymerase or aCS6 DNA polymerase, at S671, D640, Q601, and/or 1669. Exemplary CS5 DNApolymerase and CS6 DNA polymerase mutants include those comprising theamino acid substitution(s) S671F, D640G, Q601R, and/or I669F. In someembodiments, the mutant CS5 polymerase or mutant CS6 polymerasecomprises, e.g., amino acid substitutions at both D640 and S671 (e.g.,D640G and S671F). Other, exemplary CS5 DNA polymerase and CS6 DNApolymerase mutants include the following (each having the amino acidsequence of SEQ ID NO:18 or SEQ ID NO:19 except for the designatedsubstitutions):

-   -   G46E S671F;    -   G46E D640G;    -   G46E Q601R;    -   G46E I669F;    -   G46E D640G S671F;    -   G46E L329A S671F;    -   G46E L329A D640G;    -   G46E L329A Q601R;    -   G46E L329A I669F;    -   G46E L329A D640G S671F;    -   G46E S671F E678G;    -   G46E D640G E678G;    -   G46E Q601R E678G;    -   G46E I669F E678G;    -   G46E D640G S671F E678G;    -   G46E Q601R D640G S671F E678G;    -   G46E Q601R D640G S671F I669F E678G;    -   G46E L329A S671F E678G;    -   G46E L329A D640G E678G;    -   G46E L329A Q601R E678G;    -   G46E L329A I669F E678G;    -   G46E L329A D640G S671F E678G; and    -   G46E L329A Q601R D640G S671F E678G.

In addition to mutation of the motifs of SEQ ID NOS:1, 2, and/or 3 asdescribed herein, the mutant DNA polymerases of the present inventioncan also include other, non-substitutional modification(s). Suchmodifications can include, for example, covalent modifications known inthe art to confer an additional advantage in applications comprisingprimer extension. For example, in certain embodiments, the mutant DNApolymerase further includes a thermally reversible covalentmodification. In these embodiments, a modifier group is covalentlyattached to the protein, resulting in a loss of all, or nearly all, ofthe enzyme activity. The modifier group is chosen so that themodification is reversed by incubation at an elevated temperature. DNApolymerases comprising such thermally reversible modifications areparticularly suitable for hot-start applications, such as, e.g., varioushot-start PCR techniques. Thermally reversible modifier reagentsamenable to use in accordance with the mutant DNA polymerases of thepresent invention are described in, for example, U.S. Pat. No. 5,773,258to Birch et al., which is incorporated by reference herein. Exemplarymodifications include, e.g., reversible blocking of lysine residues bychemical modification of the e-amino group of lysine residues (see Birchet al., supra). In certain variations, the thermally reversible covalentmodification includes covalent attachment, to the e-amino group oflysine residues, of a dicarboxylic anhydride as described in Birch etal., supra.

For example, particularly suitable mutant polymerases comprising athermally reversible covalent modification are produced by a reaction,carried out at alkaline pH at a temperature which is less than about 25°C., of a mixture of a thermostable enzyme and a dicarboxylic acidanhydride having a general formula as set forth in the following formulaI:

where R₁ and R₂ are hydrogen or organic radicals, which may be linked;or having the following formula II:

where R₁ and R₂ are organic radicals, which may linked, and thehydrogens are cis, essentially as described in Birch et al, supra. Inspecific embodiments comprising a thermally reversible covalentmodification, the unmodified form of the polymerase is G64E CS5 DNApolymerase.

The mutant DNA polymerases of the present invention can be constructedby mutating the DNA sequences that encode the corresponding unmodifiedpolymerase (e.g., a wild-type polymerase or a corresponding variant fromwhich the mutant polymerase of the invention is derived), such as byusing techniques commonly referred to as site-directed mutagenesis.Nucleic acid molecules encoding the unmodified form of the polymerasecan be mutated by a variety of polymerase chain reaction (PCR)techniques well-known to one of ordinary skill in the art. (See, e.g.,PCR Strategies (M. A. Innis, D. H. Gelfand, and J. J. Sninsky eds.,1995, Academic Press, San Diego, Calif.) at Chapter 14; PCR Protocols: AGuide to Methods and Applications (M. A. Innis, D. H. Gelfand, J. J.Sninsky, and T. J. White eds., Academic Press, N Y, 1990).

By way of non-limiting example, the two primer system, utilized in theTransformer Site-Directed Mutagenesis kit from Clontech, may be employedfor introducing site-directed mutants into a polynucleotide encoding anunmodified form of the polymerase. Following denaturation of the targetplasmid in this system, two primers are simultaneously annealed to theplasmid; one of these primers contains the desired site-directedmutation, the other contains a mutation at another point in the plasmidresulting in elimination of a restriction site. Second strand synthesisis then carried out, tightly linking these two mutations, and theresulting plasmids are transformed into a mutS strain of E. coli.Plasmid DNA is isolated from the transformed bacteria, restricted withthe relevant restriction enzyme (thereby linearizing the unmutatedplasmids), and then retransformed into E. coli. This system allows forgeneration of mutations directly in an expression plasmid, without thenecessity of subcloning or generation of single-stranded phagemids. Thetight linkage of the two mutations and the subsequent linearization ofunmutated plasmids result in high mutation efficiency and allow minimalscreening. Following synthesis of the initial restriction site primer,this method requires the use of only one new primer type per mutationsite. Rather than prepare each positional mutant separately, a set of“designed degenerate” oligonucleotide primers can be synthesized inorder to introduce all of the desired mutations at a given sitesimultaneously. Transformants can be screened by sequencing the plasmidDNA through the mutagenized region to identify and sort mutant clones.Each mutant DNA can then be restricted and analyzed by electrophoresis,such as for example, on a Mutation Detection Enhancement gel(Mallinckrodt Baker, Inc., Phillipsburg, N.J.) to confirm that no otheralterations in the sequence have occurred (by band shift comparison tothe unmutagenized control). Alternatively, the entire DNA region can besequenced to confirm that no additional mutational events have occurredoutside of the targeted region.

Verified mutant duplexes in pET (or other) overexpression vectors can beemployed to transform E. coli such as, e.g., strain E. coli BL21 (DE3)pLysS, for high level production of the mutant protein, and purificationby standard protocols. The method of FAB-MS mapping, for example, can beemployed to rapidly check the fidelity of mutant expression. Thistechnique provides for sequencing segments throughout the whole proteinand provides the necessary confidence in the sequence assignment. In amapping experiment of this type, protein is digested with a protease(the choice will depend on the specific region to be modified since thissegment is of prime interest and the remaining map should be identicalto the map of unmutagenized protein). The set of cleavage fragments isfractionated by, for example, microbore HPLC (reversed phase or ionexchange, again depending on the specific region to be modified) toprovide several peptides in each fraction, and the molecular weights ofthe peptides are determined by standard methods, such as FAB-MS. Thedetermined mass of each fragment are then compared to the molecularweights of peptides expected from the digestion of the predictedsequence, and the correctness of the sequence quickly ascertained. Sincethis mutagenesis approach to protein modification is directed,sequencing of the altered peptide should not be necessary if the MS dataagrees with prediction. If necessary to verify a changed residue,CAD-tandem MS/MS can be employed to sequence the peptides of the mixturein question, or the target peptide can be purified for subtractive Edmandegradation or carboxypeptidase Y digestion depending on the location ofthe modification.

Mutant DNA polymerases with more than one amino acid substituted can begenerated in various ways. In the case of amino acids located closetogether in the polypeptide chain (as with amino acids X_(c4) and X_(c6)of the motif set forth in SEQ ID NO:3), they may be mutatedsimultaneously using one oligonucleotide that codes for all of thedesired amino acid substitutions. If however, the amino acids arelocated some distance from each other (separated by more than ten aminoacids, for example) it is more difficult to generate a singleoligonucleotide that encodes all of the desired changes. Instead, one oftwo alternative methods may be employed. In the first method, a separateoligonucleotide is generated for each amino acid to be substituted. Theoligonucleotides are then annealed to the single-stranded template DNAsimultaneously, and the second strand of DNA that is synthesized fromthe template will encode all of the desired amino acid substitutions. Analternative method involves two or more rounds of mutagenesis to producethe desired mutant. The first round is as described for the singlemutants: DNA encoding the unmodified polymerase is used for thetemplate, an oligonucleotide encoding the first desired amino acidsubstitution(s) is annealed to this template, and the heteroduplex DNAmolecule is then generated. The second round of mutagenesis utilizes themutated DNA produced in the first round of mutagenesis as the template.Thus, this template already contains one or more mutations. Theoligonucleotide encoding the additional desired amino acidsubstitution(s) is then annealed to this template, and the resultingstrand of DNA now encodes mutations from both the first and secondrounds of mutagenesis. This resultant DNA can be used as a template in athird round of mutagenesis, and so on. Alternatively, the multi-sitemutagenesis method of Seyfang & Jin (Anal. Biochem. 324:285-291. 2004)may be utilized.

Accordingly, also provided are recombinant nucleic acids encoding any ofthe mutant DNA polymerases of the present invention. Using a nucleicacid of the present invention, encoding a mutant DNA polymerase, avariety of vectors can be made. Any vector containing replicon andcontrol sequences that are derived from a species compatible with thehost cell can be used in the practice of the invention. Generally,expression vectors include transcriptional and translational regulatorynucleic acid regions operably linked to the nucleic acid encoding themutant DNA polymerase. The term “control sequences” refers to DNAsequences necessary for the expression of an operably linked codingsequence in a particular host organism. The control sequences that aresuitable for prokaryotes, for example, include a promoter, optionally anoperator sequence, and a ribosome binding site. In addition, the vectormay contain a Positive Retroregulatory Element (PRE) to enhance thehalf-life of the transcribed mRNA (see Gelfand et al. U.S. Pat. No.4,666,848). The transcriptional and translational regulatory nucleicacid regions will generally be appropriate to the host cell used toexpress the polymerase. Numerous types of appropriate expressionvectors, and suitable regulatory sequences are known in the art for avariety of host cells. In general, the transcriptional and translationalregulatory sequences may include, e.g., promoter sequences, ribosomalbinding sites, transcriptional start and stop sequences, translationalstart and stop sequences, and enhancer or activator sequences. Intypical embodiments, the regulatory sequences include a promoter andtranscriptional start and stop sequences. Vectors also typically includea polylinker region containing several restriction sites for insertionof foreign DNA. In certain embodiments, “fusion flags” are used tofacilitate purification and, if desired, subsequent removal of tag/flagsequence, e.g., “His-Tag”. However, these are generally unnecessary whenpurifying an thermoactive and/or thermostable protein from a mesophilichost (e.g., E. coli) where a “heat-step” may be employed. Theconstruction of suitable vectors containing DNA encoding replicationsequences, regulatory sequences, phenotypic selection genes, and themutant polymerase of interest are prepared using standard recombinantDNA procedures. Isolated plasmids, viral vectors, and DNA fragments arecleaved, tailored, and ligated together in a specific order to generatethe desired vectors, as is well-known in the art (see, e.g., Sambrook etal., Molecular Cloning: A Laboratory Manual (Cold Spring HarborLaboratory Press, New York, N.Y., 2nd ed. 1989)).

In certain embodiments, the expression vector contains a selectablemarker gene to allow the selection of transformed host cells. Selectiongenes are well known in the art and will vary with the host cell used.Suitable selection genes can include, for example, genes coding forampicillin and/or tetracycline resistance, which enables cellstransformed with these vectors to grow in the presence of theseantibiotics.

In one aspect of the present invention, a nucleic acid encoding a mutantDNA polymerase is introduced into a cell, either alone or in combinationwith a vector. By “introduced into” or grammatical equivalents herein ismeant that the nucleic acids enter the cells in a manner suitable forsubsequent integration, amplification, and/or expression of the nucleicacid. The method of introduction is largely dictated by the targetedcell type. Exemplary methods include CaPO₄ precipitation, liposomefusion, LIPOFECTIN®, electroporation, viral infection, and the like.

Prokaryotes are typically used as host cells for the initial cloningsteps of the present invention. They are particularly useful for rapidproduction of large amounts of DNA, for production of single-strandedDNA templates used for site-directed mutagenesis, for screening manymutants simultaneously, and for DNA sequencing of the mutants generated.Suitable prokaryotic host cells include E. coli K12 strain 94 (ATCC No.31,446), E. coli strain W3110 (ATCC No. 27,325), E. coli K12 strainDG116 (ATCC No. 53,606), E. coli X1776 (ATCC No. 31,537), and E. coli B;however many other strains of E. coli, such as HB101, JM101, NM522,NM538, NM539, and many other species and genera of prokaryotes includingbacilli such as Bacillus subtilis, other enterobacteriaceae such asSalmonella typhimurium or Serratia marcesans, and various Pseudomonasspecies can all be used as hosts. Prokaryotic host cells or other hostcells with rigid cell walls are typically transformed using the calciumchloride method as described in section 1.82 of Sambrook et al., supra.Alternatively, electroporation can be used for transformation of thesecells. Prokaryote transformation techniques are set forth in, forexample Dower, in Genetic Engineering, Principles and Methods 12:275-296(Plenum Publishing Corp., 1990); Hanahan et al., Meth. Enzymol., 204:63,1991. Plasmids typically used for transformation of E. coli includepBR322, pUCI8, pUCI9, pUCI18, pUC119, and Bluescript M13, all of whichare described in sections 1.12-1.20 of Sambrook et al., supra. However,many other suitable vectors are available as well.

The mutant DNA polymerases of the present invention are typicallyproduced by culturing a host cell transformed with an expression vectorcontaining a nucleic acid encoding the mutant DNA polymerase, under theappropriate conditions to induce or cause expression of the mutant DNApolymerase. Methods of culturing transformed host cells under conditionssuitable for protein expression are well-known in the art (see, e.g.,Sambrook et al., supra). Suitable host cells for production of themutant polymerases from lambda pL promotor-containing plasmid vectorsinclude E. coli strain DG116 (ATCC No. 53606) (see U.S. Pat. No.5,079,352 and Lawyer, F. C. et al., PCR Methods and Applications2:275-87, 1993, which are both incorporated herein by reference).Following expression, the mutant polymerase can be harvested andisolated. Methods for purifying the thermostable DNA polymerase aredescribed in, for example, Lawyer et al., supra.

Once purified, the ability of the mutant DNA polymerases to extendprimed templates can be tested in any of various known assays formeasuring nucleotide incorporation. For example, in the presence ofprimed template molecules (e.g., M13 DNA, etc.), an appropriate buffer,a complete set of dNTPs (e.g., dATP, dCTP, dGTP, and dTTP), and metalion, DNA polymerases will extend the primers, converting single-strandedDNA (ssDNA) to double-stranded DNA (dsDNA). This conversion can bedetected and quantified by, e.g., adding a dsDNA-binding dye, such asSYBR Green I. Using a kinetic thermocycler (see, Watson, et al. Anal.Biochem. 329:58-67, 2004, and also available from, e.g., AppliedBiosystems, Stratagene, and BioRad), digital images of reaction platescan be taken (e.g., at 10-30 second intervals), thereby allowing theprogress of the reactions to be followed. The amount of fluorescencedetected can be readily converted to extension rates. Using such routineassays, extension rates of the mutants relative to the unmodified formsof polymerase can be determined.

The mutant DNA polymerases of the present invention may be used for anypurpose in which such enzyme activity is necessary or desired.Accordingly, in another aspect of the invention, methods of primerextension using the mutant polymerases are provided. Conditions suitablefor primer extension are known in the art. (See, e.g., Sambrook et al.,supra. See also Ausubel et al., Short Protocols in Molecular Biology(4th ed., John Wiley & Sons 1999). Generally, a primer is annealed,i.e., hybridized, to a target nucleic acid to form a primer-templatecomplex. The primer-template complex is contacted with the mutant DNApolymerase and free nucleotides in a suitable environment to permit theaddition of one or more nucleotides to the 3′ end of the primer, therebyproducing an extended primer complementary to the target nucleic acid.The primer can include, e.g., one or more nucleotide analog(s). Inaddition, the free nucleotides can be conventional nucleotides,unconventional nucleotides (e.g., ribonucleotides or labelednucleotides), or a mixture thereof. In some variations, the primerextension reaction comprises amplification of a target nucleic acid.Conditions suitable for nucleic acid amplification using a DNApolymerase and a primer pair are also known in the art (e.g., PCRamplification methods). (See, e.g., Sambrook et al., supra; Ausubel etal., supra; PCR Applications: Protocols for Functional Genomics (Inniset al. eds., Academic Press 1999). In other, non-mutually exclusiveembodiments, the primer extension reaction comprises reversetranscription of an RNA template (e.g., RT-PCR). Use of the presentmutant polymerases, which provide an improved extension rate, allow for,e.g., the ability to perform such primer extension reactions withrelatively short incubation times, decreased enzyme concentrations,and/or increased product yield.

In yet other embodiments, the mutant polymerases are used for primerextension in the context of DNA sequencing, DNA labeling, or labeling ofprimer extension products. For example, DNA sequencing by the Sangerdideoxynucleotide method (Sanger et al., Proc. Natl. Acad. Sci. USA 74:5463, 1977) is improved by the present invention for polymerases capableof incorporating unconventional, chain-terminating nucleotides. Advancesin the basic Sanger et al. method have provided novel vectors(Yanisch-Perron et al., Gene 33:103-119, 1985) and base analogues (Millset al., Proc. Natl. Acad. Sci. USA 76:2232-2235, 1979; and Barr et al.,Biotechniques 4:428-432, 1986). In general, DNA sequencing requirestemplate-dependent primer extension in the presence of chain-terminatingbase analogs, resulting in a distribution of partial fragments that aresubsequently separated by size. The basic dideoxy sequencing procedureinvolves (i) annealing an oligonucleotide primer, optionally labeled, toa template; (ii) extending the primer with DNA polymerase in fourseparate reactions, each containing a mixture of unlabeled dNTPs and alimiting amount of one chain terminating agent such as a ddNTP,optionally labeled; and (iii) resolving the four sets of reactionproducts on a high-resolution denaturing polyacrylamide/urea gel. Thereaction products can be detected in the gel by autoradiography or byfluorescence detection, depending on the label used, and the image canbe examined to infer the nucleotide sequence. These methods utilize DNApolymerase such as the Klenow fragment of E. coli Pol I or a modified T7DNA polymerase.

The availability of thermostable polymerases, such as Taq DNApolymerase, resulted in improved methods for sequencing withthermostable DNA polymerase (see Innis et al., Proc. Natl. Acad. Sci.USA 85:9436, 1988) and modifications thereof referred to as “cyclesequencing” (Murray, Nuc Acids Res. 17:8889, 1989). Accordingly, mutantthermostable polymerases of the present invention can be used inconjunction with such methods. As an alternative to basic dideoxysequencing, cycle sequencing is a linear, asymmetric amplification oftarget sequences complementary to the template sequence in the presenceof chain terminators. A single cycle produces a family of extensionproducts of all possible lengths. Following denaturation of theextension reaction product from the DNA template, multiple cycles ofprimer annealing and primer extension occur in the presence ofterminators such as ddNTPs. Cycle sequencing requires less template DNAthan conventional chain-termination sequencing. Thermostable DNApolymerases have several advantages in cycle sequencing; they toleratethe stringent annealing temperatures which are required for specifichybridization of primer to nucleic acid targets as well as toleratingthe multiple cycles of high temperature denaturation which occur in eachcycle, e.g., 90-95° C. For this reason, AMPLITAQ® DNA Polymerase and itsderivatives and descendants, e.g., AmpliTaq CS DNA Polymerase andAmpliTaq FS DNA Polymerase have been included in Taq cycle sequencingkits commercialized by companies such as Perkin-Elmer (Norwalk, Conn.)and Applied Biosystems (Foster City, Calif.).

Variations of chain termination sequencing methods include dye-primersequencing and dye-terminator sequencing. In dye-primer sequencing, theddNTP terminators are unlabeled, and a labeled primer is utilized todetect extension products (Smith et al., Nature 32:674-679, 1986). Indye-terminator DNA sequencing, a DNA polymerase is used to incorporatedNTPs and fluorescently labeled ddNTPs onto the end of a DNA primer (Leeet al., Nuc. Acids. Res. 20:2471, 1992). This process offers theadvantage of not having to synthesize dye labeled primers. Furthermore,dye-terminator reactions are more convenient in that all four reactionscan be performed in the same tube.

Both dye-primer and dye-terminator methods may be automated using anautomated sequencing instrument produced by Applied Biosystems (FosterCity, Calif.) (U.S. Pat. No. 5,171,534, which is herein incorporated byreference). When using the instrument, the completed sequencing reactionmixture is fractionated on a denaturing polyacrylamide gel orcapillaries mounted in the instrument. A laser at the bottom of theinstrument detects the fluorescent products as they are electrophoresedaccording to size through the gel.

Two types of fluorescent dyes are commonly used to label the terminatorsused for dye-terminator sequencing-negatively charged and zwitterionicfluorescent dyes. Negatively charged fluorescent dyes include those ofthe fluorescein and BODIPY families. BODIPY dyes(4,4-difluoro-4-bora-3a,4a-diaza-s-indacene) are described inInternational Patent Publication WO 97/00967, which is incorporatedherein by reference. Zwitterionic fluorescent dyes include those of therhodamine family. Commercially available cycle sequencing kits useterminators labeled with rhodamine derivatives. However, therhodamine-labeled terminators are rather costly and the product must beseparated from unincorporated dye-ddNTPs before loading on the gel sincethey co-migrate with the sequencing products. Rhodamine dye familyterminators seem to stabilize hairpin structures in GC-rich regions,which causes the products to migrate anomalously. This requires the useof dITP, which relaxes the secondary structure but also affects theefficiency of incorporation of terminator.

In contrast, fluorescein-labeled terminators eliminate the separationstep prior to gel loading since they have a greater net negative chargeand migrate faster than the sequencing products. In addition,fluorescein-labeled sequencing products have better electrophoreticmigration than sequencing products labeled with rhodamine. Althoughwild-type Taq DNA polymerase does not efficiently incorporateterminators labeled with fluorescein family dyes, this can now beaccomplished efficiently by use of the modified enzymes as described inU.S. Patent Application Publication No. 2002/0142333, which isincorporated by reference herein in its entirety. Accordingly,modifications as described in US 2002/0142333 can be used in the contextof the present invention to produce fluorescein-family-dye-incorporatingthermostable polymerases having improved primer extension rates. Forexample, in certain embodiments, the unmodified DNA polymerase inaccordance with the present invention is a modified thermostablepolymerase as described in US 2002/0142333 and having the motifs setforth in SEQ ID NOS:1, 2, and 3.

Other exemplary nucleic acid sequencing formats in which the mutant DNApolymerases of the invention can be used include those involvingterminator compounds that include 2′-PO₄ analogs of ribonucleotides(see, e.g., U.S. Application Publication Nos. 2005/0037991,2005/0037398, 2007/0219361 and 2007/0154914, which are each incorporatedby reference). The mutant DNA polymerases described herein generallyimprove these sequencing methods, e.g., by reducing the time necessaryfor the cycled extension reactions and/or by reducing the amount orconcentration of enzyme that is utilized for satisfactory performance.

In another aspect of the present invention, kits are provided for use inprimer extension methods described herein. Typically, the kit iscompartmentalized for ease of use and contains at least one containerproviding a mutant DNA polymerase in accordance with the presentinvention. One or more additional containers providing additionalreagent(s) can also be included. Such additional containers can includeany reagents or other elements recognized by the skilled artisan for usein primer extension procedures in accordance with the methods describedabove, including reagents for use in, e.g., nucleic acid amplificationprocedures (e.g., PCR, RT-PCR), DNA sequencing procedures, or DNAlabeling procedures. For example, in certain embodiments, the kitfurther includes a container providing a 5′ sense primer hybridizable,under primer extension conditions, to a predetermined polynucleotidetemplate, or a primer pair comprising the 5′ sense primer and acorresponding 3′ antisense primer. In other, non-mutually exclusivevariations, the kit includes one or more containers providing freenucleotides (conventional and/or unconventional). In specificembodiments, the kit includes alpha-phophorothioate dNTPs, dUTP, dITP,and/or labeled dNTPs such as, e.g., fluorescein- or cyanin-dye familydNTPs. In still other, non-mutually exclusive embodiments, the kitincludes one or more containers providing a buffer suitable for a primerextension reaction.

Examples

It is understood that the examples and embodiments described herein arefor illustrative purposes only and are not intended to limit the scopeof the claimed invention. It is also understood that variousmodifications or changes in light the examples and embodiments describedherein will be suggested to persons skilled in the art and are to beincluded within the spirit and purview of this application and scope ofthe appended claims.

Example I: Identification and Characterization of Mutant DNA Polymeraseswith Improved Enzyme Activity

Mutations in CS family polymerases were identified that provide, e.g.,improved ability to extend primed DNA templates in the presence of freenucleotides. In brief, the steps in this screening process includedlibrary generation, expression and partial purification of the mutantenzymes, screening of the enzymes for the desired property, DNAsequencing, clonal purification, and further characterization ofselected candidate mutants, and generation, purification, andcharacterization of combinations of the mutations from the selectedmutants. Each of these steps is described further below.

The mutations identified by this process include S671F, D640G, Q601R,and I669F, either separately or in various combinations. These mutationswere placed in several CS-family polymerases, including G46E CS5, G46EL329A CS5, G46E E678G CS5, and G46E L329A E678G CS5. Some of thesemutant polymerases are listed in Table 2. Other exemplary mutantpolymerases that have been made include CS6 G46E Q601R D640G S671F E678GDNA polymerase and certain Thermus sp. Z05 DNA polymerase mutants. Theresulting mutant polymerases were characterized by analyzing theirperformance in a series of Kinetic Thermal Cycling (KTC) experiments.

TABLE 2 Exemplary CS5 DNA Polymerase Mutants G46E D640G G46E S671F E678GG46E S671F G46E D640G S671F E678G G46E Q601R D640G G46E Q601R D640GS671F E678G G46E D640G S671F G46E L329A Q601R E678G G46E Q601R D640GS671F G46E L329A D640G E678G G46E L329A D640G G46E L329A S671F E678GG46E L329A Q601R D640G G46E L329A Q601R S671F E678G S671F G46E L329AS671F G46E L329A D640G S671F E678G G46E L329A Q601R D640G G46E L329AQ601R D640G S671F E678G G46E L329A D640G S671F G46E L329A D640G I669FS671F E678G L329A D640G L329A Q601R E678G L329A D640G S671F L329A S671FE678G L329A Q601R D640G S671F S671F L329A S671F D640G S671F D640G Q601RD640G S671F

The identified mutations, S671F, D640G, Q601R, and I669F, resulted in,e.g., an improved ability to extend primed templates. In the particularcontext of the E678G mutation, which allows for the incorporation ofribonucleotides and other 2′-modified nucleotides, but which alsoresults in an impaired ability to extend primed templates, the S671F,D640G, Q601R, and I669F mutations ameliorated this property of impairedprimer extension ability. The identified mutations, particularly S671Falone and S671F plus D640G, also showed improved efficiency of reversetranscription when placed in G46E CS5 and G46E L329A CS5 DNApolymerases. Additional features of the mutant DNA polymerases of theinvention are described further below.

Clonal Library Generation:

A nucleic acid encoding the polymerase domain of CS5 E678G DNApolymerase was subjected to error-prone (mutagenic) PCR between Bgl IIand Hind III restriction sites of a plasmid including this nucleic acidsequence. PCR was performed using a range of Mg⁺² concentrations from1.8-3.5 mM, in order to generate libraries with a corresponding range ofmutation rates. Buffer conditions were: 50 mM Bicine pH 8.2, 115 mMKOAc, 8% w/v glycerol, 0.2 mM each dNTPs, and 0.2×SYBR Green I. AGeneAmp® AccuRT Hot Start PCR enzyme was used at 0.15 U/μl. Startingwith 5×10⁵ copies of linearized CS5 E678G plasmid DNA/reaction volume of50 μl, 30 cycles of amplification were performed, using an annealingtemperature of 60° C. for 15 seconds, an extension temperature of 72° C.for 45 seconds, and a denaturation temperature of 95° C. for 15 seconds.

The resulting amplicon was purified over a Qiaquick spin column (Qiagen,Inc., Valencia, Calif., USA) and cut with Bgl II and Hind III, thenre-purified. A vector plasmid, a modification of G46E L329A CS5 carryinga large deletion in the polymerase domain between the BglII and HindIIIsites, was prepared by cutting with the same two restriction enzymes andtreating with calf intestinal phosphatase (CIP). The cut vector and themutated insert were mixed at different ratios and treated with T4 ligaseovernight at 15° C. The ligations were purified and transformed into anE. coli host strain by electroporation.

Aliquots of the expressed cultures were plated on ampicillin-selectivemedium in order to determine the number of unique transformants in eachtransformation. Transformations with the most unique transformants ateach mutagenesis rate were stored at −70 to −80° C. in the presence ofglycerol as a cryo-protectant.

Each library was then spread on large format ampicillin-selective agarplates. Individual colonies were transferred to 384-well platescontaining 2× Luria broth with ampicillin and 10% w/v glycerol using anautomated colony picker (QPix2, Genetix Ltd). These plates wereincubated overnight at 30° C. to allow the cultures to grow, then storedat −70 to −80° C. The glycerol added to the 2× Luria broth was lowenough to permit culture growth and yet high enough to providecryo-protection. Several thousand colonies at several mutagenesis (Mg⁺²)levels were prepared in this way for later use.

Extract Library Preparation Part 1—Fermentation:

From the clonal libraries described above, a corresponding library ofpartially purified extracts suitable for screening purposes wasprepared. The first step of this process was to make small-scaleexpression cultures of each clone. These cultures were grown in 96-wellformat; therefore there were 4 expression culture plates for each384-well library plate. One μl was transferred from each well of theclonal library plate to a well of a 96 well seed plate, containing 150μl of Medium A (see Table 3 below). This seed plate was shaken overnightat 1150 rpm at 30° C., in an iEMS plate incubater/shaker(ThermoElectron). These seed cultures were then used to inoculate thesame medium, this time inoculating 10 μl into 300 μl Medium A in largeformat 96 well plates (Nunc #267334). These plates were incubatedovernight at 37° C. The expression plasmid contained transcriptionalcontrol elements, which allow for expression at 37° C. but not at 30° C.After overnight incubation, the cultures expressed the clone protein attypically 1-10% of total cell protein. The cells from these cultureswere harvested by centrifugation. These cells were either frozen (−20°C.) or processed immediately, as described below.

TABLE 3 Medium A (Filter-sterilized prior to use) ComponentConcentration MgSO₄•7H₂O 0.2 g/L Citric acid•H₂O 2 g/L K₂HPO₄ 10 g/LNaNH₄PO₄•4H₂O 3.5 g/L MgSO₄ 2 mM Casamino acids 2.5 g/L Glucose 2 g/LThiamine•HCl 10 mg/L Ampicillin 100 mg/L

Extract Library Preparation Part 2—Extraction:

Cell pellets from the fermentation step were resuspended in 30 μl Lysisbuffer (Table 4 below) and transferred to 384-well thermocycler plates.Note that the buffer contained lysozyme to assist in cell lysis, and twonucleases to remove both RNA and DNA from the extract. The plates weresubjected to three rounds of freeze-thaw (−70° C. freeze, 37° C. thaw,not less than 15 minutes per step) to lyse the cells. Ammonium sulfatewas added (5 μl of a 0.75M solution) and the plates incubated at 75° C.for 15 minutes in order to precipitate and inactivate contaminatingproteins, including the exogenously added nucleases. The plates werecentrifuged at 3000×g for 15 minutes and the supernatants transferred toa fresh 384-well thermocycler plate. These extract plates were frozen at−20° C. for later use in screens. Each well contained about 0.5-3 μM ofthe mutant library polymerase enzyme.

TABLE 4 Lysis Buffer Component Concentration or Percentage Tris pH 8.020 mM EDTA 1 mM MgCl₂ 5 mM TLCK 1 mM Leupeptin 1 μg/ml Pefabloc 0.5mg/ml Tween 20 0.5% v/v Lysozyme (from powder) 2 mg/ml RNase 0.025 mg/mlDNase I 0.075 Units/μl

Screening Extract Libraries for Improved Extension Rate:

M13mp18 single-stranded DNA (M13; GenBank Accession No. X02513), primedwith an oligonucleotide having the following sequence:

(SEQ ID NO: 72) 5′-GGGAAGGGCGATCGGTGCGGGCCTCTTCGC-3′was used as the template molecule in the extension assay screen. 0.5-1.0μl of extract was added to 10-20 μl reaction master mix containing 0.5-1nM primed M13 template in 384-well PCR plates. Extension of the primedtemplate was monitored every 10-30 seconds in a modified kinetic thermalcycler using a CCD camera (see, Watson, supra). A typical reactionmaster mix is listed below. Master mixes invariably included metal ion,usually magnesium at 1-4 mM, a mixture of all four dNTPs or dNTPanalogs, buffer components to control the pH and the ionic strength,typically 25 mM Tricine pH 8.3/35 mM KOAc, and SYBR Green I at 0.6×(Molecular Probes), which allowed for the fluorescent detection ofprimer strand extension. In order to distinguish extension-derivedfluorescence from background fluorescence, parallel wells were includedin the experiment in which primer strand extension was prevented, forexample, by adding a metal chelator such as EDTA, or leaving out thenucleotides from the reaction master mix.

In order to find mutant enzymes that have improved nucleic acidextension rates in the presence of ribonucleotides, extension reactionswere run in the presence and absence of ribonucleotides and theresulting rates of extension were compared, using the methods describedabove. Adding a high level of ribonucleotide (for example, a 50:50 mixof rATP and dATP) reduced the rate of extension of the parental enzyme,G46E L329A E678G CS5. Mutant extracts that exhibited a reduced level ofinhibition by ribonucleotides were identified in this screen. Primaryscreening was done on the scale of thousands of extracts. The topseveral percent of these were chosen for re-screening. Culture wellscorresponding to the top extracts were sampled to fresh growth mediumand re-grown to produce a new culture plate containing all of the topproducers, as well as a number of parental cultures to be used forcomparison. These culture plates were then fed into the same screeningprocess, to get more data on the candidate mutants. Following thissecondary screening round, a relatively small number of extracts stillappeared to consistently display improved extension rate relative to theparental clone. These clones were chosen for further testing. They werefirst streaked on selective agar plates to ensure clonal purity, thenthe DNA sequence of the mutated region of the polymerase gene wassequenced to determine the mutation(s) that were present in any singleclone. In parallel with this work, enough mutant enzyme was produced inshake flask culture for the concentration to be determined by gel-baseddensitometry, after partial purification in a manner similar to thatused to prepare the primary extracts. These quantified extracts werecompared to parental enzyme in the conditions used in the screen, but atequal protein concentration. This final screen ensured that thedifferences observed were not simply protein concentration effects.

Following this final round of screening, four clones still appeared tohave improved extension rates in the presence of ribonucleotides. Thesequences of these four clones were determined to code for the followingamino acid changes relative to the parental strain:

-   -   clone 1: S553T D640G D664G E830A    -   clone 2: S671F    -   clone 3: F557L I669F    -   clone 4: Q601R Y739C V749A        In the case of clone 2, it was clear that the S671F mutation        must have been responsible for the observed phenotype, since it        was the only amino acid mutation in the clone. For the other        three clones, it was initially impossible to tell which        mutation, or combination of mutations, was responsible for the        observed phenotype. Therefore, the individual mutations were        separated from one another, by combining DNA from the mutant        plasmid with the parental plasmid using restriction fragment        swaps. This is easily effected in cases where a vector-unique        restriction site exists between mutations to be separated. For        clone 1, such sites exist between all four of the mutations,        accordingly it was possible to prepare plasmids containing each        mutation individually as well as other plasmids carrying any 2        or 3 of the 4 original mutations. For clone 4, there was no such        site between Y739C and V749A, but there was a site between Q601R        and Y739C. Therefore it was possible to prepare plasmid DNA        encoding a polymerase carrying just the Q601R mutation, and        another plasmid carrying the Y739C/V749A combination.

These new plasmids were transformed into the E. coli host, andpolymerase protein was expressed, purified to homogeneity, andquantified. These resulting new mutant enzymes were compared to theparental types and to the original mutant enzymes under the conditionsof the original screen. It was clear from this data that the mutationD640G was solely responsible for the improved phenotype of mutant clone1, that the mutation I669F was responsible for the improvements inmutant clone 3, and that the mutation Q601R was responsible for theimprovements in mutant clone 4.

These active mutations were then combined with one another, and movedinto different CS-type backbones (see, e.g., Table 2, above), againusing restriction fragment swaps to create the desired expressionplasmid, then transforming the plasmid into the E. coli host, andfinally expressing, purifying to homogeneity, and quantifying the mutantpolymerase, as described above. These new combination mutants weretested for the ability to extend primed M13 DNA in the presence ofribonucleotides. Interestingly, it was found that combining themutations D640G, S671F, and Q601R resulted in an increase in extensionrate relative to clones carrying only a single mutation. The doublecombination mutants tested, including D640G S671F and Q601R S671F, alsoshowed improved extension rates relative to strains carrying only asingle mutation. Moreover, the combination mutants also demonstratedimproved rates of extension on primed M13 DNA when only dNTPs werepresent, when compared to the parental type, and furthermore it wasobserved that the degree of improvement relative to the parental typewas greatest when the extension rate experiment was performed at lowenzyme concentration or relatively high salt concentration. Theseobservations were repeated when the combination mutations were movedinto a genetic backbone that did not include the riboincorporatingmutation E678G. Surprisingly, even in the E678 background, theindividual mutant enzymes and the combination mutant enzymes were even“faster” than their corresponding E678 “parents.” These and othercharacteristics of the mutant polymerases of the invention are furtherillustrated in the examples provided below.

Example II: Properties of Mutants of G46E L329A E678G CS5 DNA PolymeraseUnder Varied Salt Concentrations

The nucleic acid extension rates of various mutants of G46E L329A E678GCS5 DNA polymerase were determined in the presence of 90% riboadenosinetriphosphate (ribo ATP or rATP). The reaction mixture contained 25 mMTricine pH 8.3, 20 mM (FIG. 4) or 60 mM (FIG. 5) KOAc, 3 mM MgCl₂, 2.5%v/v Storage Buffer (50% v/v glycerol, 100 mM KCl, 20 mM Tris pH 8.0, 0.1mM EDTA, 1 mM DTT, 0.5% Tween 20), 1% DMSO, 1×SYBR Green I, 0.5 nMprimed M13, and 5 nM enzyme. To this, nucleotides were added to a finalconcentration of 0.1 mM dGTP, 0.1 mM dTTP, and 0.1 mM dCTP, 0.01 mMdATP, and 0.09 mM ribo ATP. Parallel reactions containing no nucleotideswere also set up. All reactions were run in quadruplicate in 20 μlvolume in 384 well thermocycler plates. The extension of primed M13template was monitored by fluorescence in a kinetic thermocycler set at64° C., taking readings every 10 seconds. Replicate identical reactionswere averaged and the parallel minus nucleotide reactions subtracted.Extension rate was estimated by linear regression analysis of theresulting data.

As indicated above, FIGS. 4 and 5 show results obtained from theseanalyses. For example, FIGS. 4 and 5 illustrate that improved nucleicacid extension rates result from various mutants described herein, whenribonucleotides are present in reaction mixtures and incorporated on aDNA template. As further shown, for example, when certain mutations arecombined in a single mutant enzyme, even further extension rateimprovements are observed.

Example III: Properties of Mutants of G46E L329A CS5 DNA PolymeraseUnder Varied Salt Concentrations

The nucleic acid extension rate of various mutants of G46E L329A CS5 DNApolymerase, as well as Thermus sp. Z05 DNA polymerase and its truncate,delta Z05 DNA polymerase (see, e.g., U.S. Pat. No. 5,455,170, entitled“MUTATED THERMOSTABLE NUCLEIC ACID POLYMERASE ENZYME FROM THERMUSSPECIES Z05” issued Oct. 3, 1995 to Abramson et al. and U.S. Pat. No.5,674,738, entitled “DNA ENCODING THERMOSTABLE NUCLEIC ACID POLYMERASEENZYME FROM THERMUS SPECIES Z05” issued Oct. 7, 1997 to Abramson et al.,which are both incorporated by reference), was determined. The reactionmixture contained 25 mM Tricine pH 8.3, 0 mM (FIG. 6) or 60 mM (FIG. 7)KOAc, 3 mM MgCl₂, 2.5% v/v Storage Buffer (50% v/v glycerol, 100 mM KCl,20 mM Tris pH 8.0, 0.1 mM EDTA, 1 mM DTT, 0.5% Tween 20), 1% DMSO,1×SYBR Green I, 0.5 nM primed M13, and 5 nM enzyme. To this, nucleotideswere added to a final concentration of 0.1 mM dGTP, 0.1 mM dTTP, and 0.1mM dCTP, and 0.1 mM dATP. Parallel reactions containing no nucleotideswere also set up. All reactions were run in quadruplicate in 20 μlvolume in 384 well thermocycler plates. The extension of primed M13template was monitored by fluorescence in a kinetic thermocycler set at64° C., taking readings every 10 seconds. Replicate identical reactionswere averaged and the parallel minus nucleotide reactions subtracted.Extension rate was estimated by linear regression analysis of theresulting data.

The data shown in FIGS. 6 and 7 illustrate, e.g., that certain mutationsdescribed herein result in improved nucleic acid extension rates evenwhen ribonucleotides are not present in the reaction mixtures, and evenin a genetic backbone that does not include theribonucleotide-incorporation mutation, E678G. As further shown, forexample, this rate improvement is even greater when the mutations arecombined in a single mutant enzyme.

Example IV: Effect of Salt Concentration on the Extension Rates ofVarious Mutant CS5 DNA Polymerases

The nucleic acid extension rate of various mutants of G46E L329A CS5 DNApolymerase, as well as Thermus sp. Z05 DNA polymerase and its truncate,delta Z05 DNA polymerase, was determined. The reaction mixture contained25 mM Tricine pH 8.3, 0-100 mM KOAc, 3 mM MgCl₂, 2.5% v/v Storage Buffer(50% v/v glycerol, 100 mM KCl, 20 mM Tris pH 8.0, 0.1 mM EDTA, 1 mM DTT,0.5% Tween 20), 1% DMSO, 1×SYBR Green I, 0.5 nM primed M13, and 25 nM(FIG. 8) or 5 nM (FIG. 9) enzyme. To this, nucleotides were added to afinal concentration of 0.1 mM dGTP, 0.1 mM dTTP, and 0.1 mM dCTP, and0.1 mM dATP. Parallel reactions containing no nucleotides were also setup. All reactions were run in quadruplicate in 20 μl volume in 384 wellthermocycler plates. The extension of primed M13 template was monitoredby fluorescence in a kinetic thermocycler set at 64° C., taking readingsevery 10 seconds. Replicate identical reactions were averaged and theparallel minus nucleotide reactions subtracted. Extension rate wasestimated by linear regression analysis of the resulting data.

The data shown in FIGS. 6 and 7 illustrate, among other properties,e.g., that the increased nucleic acid extension rates conferred by thecertain mutants described herein are maintained over a wide range ofsalt and enzyme concentrations, and also that the mutations confer anextension rate increase in a genetic background that includes fullproof-reading activity.

Example V: Use of Various Mutant CS5 DNA Polymerases in RT-PCR

Mg²⁺-Based RT:

The mutations Q601R, D640G, and S671F, separately and in combination,were evaluated for their effect on PCR and RT-PCR efficiency in thepresence of Mg⁺². The reactions all contained the following components:50 mM Tricine pH 8.0, 2.5 mM Mg(OAc)₂, 6% v/v Storage Buffer (50% v/vglycerol, 100 mM KCl, 20 mM Tris pH 8.0, 0.1 mM EDTA, 1 mM DTT, 0.2%Tween 20), 0.2×SYBR Green I, 0.02 units/μl UNG, 0.2 mM each dATP, dCTP,and dGTP, 0.3 mM dUTP, 0.03 mM dTTP, and 200 nM of each primer, whereinthe primers comprise a 2′-amino-C at the 3′-end at the 3′-end.

Enzymes were used at their pre-determined concentration and KOAc optima.These are given in Table 5.

TABLE 5 Pol KOAc Pol KOAc Polymerase (nM) (mM) Polymerase (nM) (mM) G236 25 GL 236 25 GD 59 50 GLD 59 50 GS 118 25 GLS 118 25 GDS 23.6 25GLDS 23.6 25 GQDS 23.6 100 GLQDS 23.6 100

Each enzyme was tested with both 10⁶ copies/50 μl reaction DNA template(pAW109 plasmid DNA) and 10⁶ copies/50 μl reaction RNA template (pAW109transcript). Reactions were run in a kinetic thermocycler (ABI 5700thermalcycler). The thermocycling parameters were: 50° C. for 2 minutes;65° C. for 45 minutes; 93° C. for 1 minute; then 40 cycles of: 93° C.for 15 seconds; and 65° C. for 30 seconds. Fluorescence data wasanalyzed to determine Ct values (emergence of fluorescence overbaseline) (FIG. 10). More specifically, the data shown in FIG. 10 (seealso, FIG. 11) illustrates, among other properties, e.g., that themutations described herein, either singly or in combination, improve theefficiency of the Mg²⁺-activated reverse transcription activity of themutant enzyme relative to the corresponding parent or non-mutant enzyme.For example, the GLDS enzyme performed well, e.g., when the time allowedfor reverse transcription was decreased to 5 minutes, as shown in FIG.11 (referred to additionally below).

Mg²⁺-Based RT with Reduced RT Time:

The mutations Q601R, D640G, and S671F, separately and in combination,were evaluated for their effect on RT-PCR efficiency in the presence ofMg⁺², using either 45 minute or 5 minute RT time. The reactions allcontained the following components: 50 mM Tricine pH 8.0, 2.5 mMMg(OAc)₂, 6% v/v Storage Buffer (50% v/v glycerol, 100 mM KCl, 20 mMTris pH 8.0, 0.1 mM EDTA, 1 mM DTT, 0.2% Tween 20), 1% DMSO, 0.2×SYBRGreen I, 0.02 units/μl UNG, 0.2 mM each dATP, dCTP, and dGTP, 0.3 mMdUTP, 0.03 mM dTTP, and 200 nM of each primer, wherein the primerscomprise a 2′-amino-C at the 3′-end.

Enzymes were used at their pre-determined concentration and KOAc optima.These are given in the following Tables 6 and 7:

TABLE 6 45 minute RT Time: Pol KOAc Pol KOAc Polymerase (nM) (mM)Polymerase (nM) (mM) G 236 25 GL 236 25 GD 59 50 GLD 59 50 GS 118 25 GLS118 25 GDS 23.6 25 GLDS 23.6 25 GQDS 23.6 100 GLQDS 23.6 100

TABLE 7 5 minute RT time: Pol KOAc Pol KOAc Polymerase (nM) (mM)Polymerase (nM) (mM) G 118 25 GL 236 55 GD ~ ~ GLD 94.4 50 GS 118 25 GLS118 25 GDS 23.6 25 GLDS 106.2 50 GQDS ~ ~ GLQDS 23.6 100 ~ denotescondition that was not done

Each enzyme was tested with 10⁶ copies/50 μl reaction RNA template(pAW109 transcript). Reactions were run in a kinetic thermocycler(ABI5700). The thermocycling parameters were: 50° C. for 2 minutes; 65°C. for 5 minutes or 45 minutes; 93° C. for 1 minute; then 40 cycles of:93° C. for 15 seconds; and 65° C. for 30 seconds. Fluorescence data wasanalyzed to determine Ct values (emergence of fluorescence overbaseline) (FIG. 11).

Mn²⁺-Based RT with Reduced RT Time:

The mutations Q601R, D640G, and S671F, separately and in combination,were evaluated for their effect on RT-PCR efficiency in the presence ofMn⁺², using either 45 minute or 5 minute RT time. The reactions allcontained the following components: 50 mM Tricine pH 8.0, 1 mM Mn(OAc)₂,6% v/v Storage Buffer (50% v/v glycerol, 100 mM KCl, 20 mM Tris pH 8.0,0.1 mM EDTA, 1 mM DTT, 0.2% Tween 20), 1% DMSO, 0.2×SYBR Green I, 0.02units/μl UNG, 0.2 mM each dATP, dCTP, and dGTP, 0.3 mM dUTP, 0.03 mMdTTP, and 200 nM of each primer, wherein the primers comprise a2′-amino-C at the 3′-end.

Enzymes were used at their pre-determined concentration/KOAc optima.These are given in the following Tables 8 and 9:

TABLE 8 45 minute RT Time: Pol KOAc Pol KOAc Polymerase (nM) (mM)Polymerase (nM) (mM) G 236 55 GL 236 55 GD ~ ~ GLD 59 55 GS 118 55 GLS118 55 GDS 23.6 55 GLDS 23.6 70 GQDS 23.6 100 GLQDS 23.6 100

TABLE 9 5 minute RT time: Pol KOAc Pol KOAc Polymerase (nM) (mM)Polymerase (nM) (mM) G ~ ~ GL 354 68 GD ~ ~ GLD ~ ~ GS ~ ~ GLS 59 55 GDS~ ~ GLDS 23.6 70 GQDS 59 100 GLQDS 11.8 100 ~ denotes condition that wasnot done

Each enzyme was tested with 10⁵ copies/50 μl reaction RNA template(pAW109 transcript). Reactions were run in a kinetic thermocycler(ABI5700). The thermocycling parameters were: 50° C. for 2 minutes; 65°C. for 5 minutes or 45 minutes; 93° C. for 1 minute; then 40 cycles of:93° C. for 15 seconds; and 65° C. for 30 seconds. Fluorescence data wasanalyzed to determine Ct values (emergence of fluorescence overbaseline) (FIG. 12). More specifically, the data shown in FIG. 12illustrates, among other properties, e.g., that improved Mn²⁺-activatedreverse transcription efficiency results from certain of the mutationsdescribed herein, either singly or in combination, and that thisimprovement is enhanced when the time allowed for reverse transcriptionis decreased.

Example VI: Fragmentation Using Low-Level Ribonucleoside TriphosphateIncorporation

It is sometimes useful to fragment a PCR product, for example whenanalyzing the product in a hybridization-based assay. Fragmentation canbe easily accomplished by treating with alkali and heat, ifribonucleotides have been incorporated into the PCR product. For suchapplications relatively low level ribo-substitution will suffice toachieve fragments of optimal length. The ability of various mutant DNApolymerases to generate ribo-substituted PCR product of length 1 kb wasdemonstrated in the following example.

The reaction mixture was composed of 100 mM Tricine pH 8.3, 75 mM KOAc,5% v/v glycerol, 2.5 mM Mg(OAc)₂, 50 nM enzyme, 0.1% v/v DMSO, and 2.5%v/v enzyme storage buffer (50% v/v glycerol, 100 mM KCl, 20 mM Tris pH8.0, 0.1 mM EDTA, 1 mM DTT, 0.5% Tween 20). Various mixtures of dNTPsand rNTPS were tested. In all cases, the sum of rATP and dATP was 200μM, as was the sum of dCTP and rCTP, and dGTP and rGTP. The sum of dTTPand rTTP was 40 μM and the sum of dUTP and rUTP was 360 μM. In thisanalysis either all four rNTPS were added together, up to 10% of thetotal (see, “rNTP Series” in FIGS. 13A and 13B (% rNTP indicated abovethe relevant lane in the gel)), or rATP alone was added, up to 50% ofthe total (see, “rATP Series” in FIGS. 13A and 13B (% rATP indicatedabove the relevant lane in the gel)). Enzymes tested were GQDSE,CS6-GQDSE, GLQDSE, GDSE, GLDSE, GLDE, GE, and a 4:1 mixture of GL andGLE (G=G46E, L=L329A, Q=Q601R, D=D640G, S=S671F, and E=E678G).

This reaction mix included primers used to generate a 1 kb product froman M13 template. The primers were used at 200 nM each, wherein theprimers comprise a 2′-amino-C at the 3′-end. M13 DNA was added to 10⁶copies per 100 μl reaction.

Reactions were run in an ABI 9700 thermocycler. The thermocyclingparameters were: 50° C. for 15 seconds; 92° C. for 1 minute; then 30cycles of: 92° C. for 15 seconds; followed by an extension step of 62°C. for 4 minutes. The ability to make full length amplicon under thevarious conditions tested was determined by agarose gel electrophoresis,loading 5 μl of each reaction per lane on a 2% egel-48 (Invitrogen)(FIGS. 13A and 13B). More specifically, these figures show, e.g., thatcertain mutant enzymes described herein are able to produce full-length(1 kb) amplicons at higher levels of ribonucleotides present in thereaction mixtures than the corresponding parental or non-mutant G46ECSSR enzyme. For example, the mixture of GL CS5 and GLE enzymes madeamplicon at the highest level of ribonucleotide assayed in this example,but because GL CS5 polymerase cannot incorporate ribonucleotides, theseamplicons contained a relatively low level of ribonucleotidesincorporated in the amplicon.

These amplicons were then fragmented as follows: 2 μl amplicon wasdiluted 27.5× in 0.3N NaOH and 20 mM EDTA, then heated at 98° C. for 10minutes. The fragmented amplicon was neutralized by adding 2.5 μl 6 NHCl. To determine the degree of fragmentation achieved, the copy numberof an internal fragment of the amplicon was compared before and afterfragmentation, using quantitative PCR without UNG. The cycle delayobserved due to fragmentation is an indication of the degree offragmentation (and of ribonucleotide incorporation). Increasedribonucleotide incorporation leads to increased Ct delay. For thisamplification, the reaction mixture was composed of 100 mM Tricine pH8.3, 50 mM KOAc, 5% v/v glycerol, 2.5 mM Mg(OAc)₂, 20 nM GQDS, 0.5%DMSO, 0.1×SYBR Green I, 2.5% v/v enzyme storage buffer (50% v/vglycerol, 100 mM KCl, 20 mM Tris pH 8.0, 0.1 mM EDTA, 1 mM DTT, 0.5%Tween 20), 200 μM each dCTP, dGTP, and dATP, 360 μM dUTP, and 40 μMdTTP. This reaction mix was used to generate a 340 bp product from thefragmented and unfragmented amplicons, diluting these templates afurther 10,000-fold from the dilution used for fragmentation. The primersequences were used at 200 nM each, wherein the primers comprise a2′-amino-C at the 3′-end.

Reactions were run in 384-well plates, 20 μl per reaction in a kineticthermocycler. The thermocycling parameters were: 50° C. for 15 seconds;92° C. for 1 minute; then 46 cycles of: 92° C. for 15 seconds; followedby an extension step of 62° C. for 1 minute. Threshold Cts weredetermined and corresponding fragmented and unfragmented Cts werecompared, thus generating a delta Ct for each enzyme/rNTP conditiontested. In this example, the greater the amount of incorporated NTP(reflecting an improved ability to incorporate NTPs in the presence ofdNTPs), the greater will be the delta Ct or Ct delay afteralkali-induced fragmentation. These are shown in FIGS. 14A and 14B. Thedata show, e.g., that the mutant enzymes of the invention are superiorin the incorporation of ATP or NTP in generating PCR products that haveincreased extents of ribonucleotide substitution. Compare any of theillustrated enzymes to the parental blend “GL/GLE” or to “C5R”.Increased fragmentation derives from increased ribonucleotideincorporation and an improved ability to incorporate a limitingconcentration of ribonucleotides in the presence deoxynucleotides.

Hybridization assays frequently involve attaching biotin to the moleculebeing detected. It is therefore useful to incorporate biotin into PCRproduct. If biotin is attached to ribonucleotide, each fragment (exceptthe 3′ most distal fragment, which is usually complementary to the otherprimer and therefore uninformative) will carry a single biotin moiety,which will result in equal signal generation by each fragment.

The ability of various enzymes to incorporate ribo-nucleotides linked toa biotin into PCR product was determined, as described below. Thereaction mixture was composed of 100 mM Tricine pH 8.3, 75 mM KOAc, 5%v/v glycerol, 2.5 mM Mg(OAc)₂, 50 nM enzyme, 0.1% DMSO, 2.5% v/v enzymestorage buffer (50% v/v glycerol, 100 mM KCl, 20 mM Tris pH 8.0, 0.1 mMEDTA, 1 mM DTT, 0.5% Tween 20), 200 μM each dCTP+analogs, dGTP, anddATP, 360 μM dUTP, and 40 μM dTTP. rCTP up to 40% of the total orbiotin-LC-rCTP up to 50% of the total were tested. Enzymes (CS5polymerases) tested were GE, GQDSE, GDSE, and a 4:1 blend of GL and GLE(G=G46E, L=L329A, Q=Q601R, D=D640G, S=S671F, and E=E678G).

This reaction mix was used to generate a 1 kb product from an M13template, using primer sequences comprising a 2′-amino-C at 200 nM each.M13 DNA was added to 5×10⁵ copies per 50 μl reaction. Reactions were runin an ABI 9700 thermocycler. The thermocycling parameters were: 50° C.for 15 seconds; 92° C. for 1 minute; then 30 cycles of: 92° C. for 15seconds; followed by an extension step of 62° C. for 4 minutes. Theability to make full length amplicon under the various conditions testedwas determined by agarose gel electrophoresis, loading 5 μl of eachreaction per lane on a 2% egel-48 (Invitrogen) (FIGS. 15A and 15B). Morespecifically, FIGS. 15A and 15B show, e.g., that mutants GQDSE and GDSEare both able to produce amplicon in higher levels of rCTP andbiotinylated rCTP than can the corresponding parental or non-mutant G46ECSSR enzyme. Further while the GL/GLE blend can produce amplicon, thisamplicon will have a low level of either rCTP or biotinylated rCTPincorporation, because the GL enzyme cannot incorporate these compounds.

These amplicons were then fragmented as follows: 2 μl amplicon wasdiluted 27.5× in 0.3N NaOH and 20 mM EDTA, then heated at 98° C. for 10minutes. The fragmented amplicon was neutralized by adding 2.5 μl 6 NHCl. To determine the degree of fragmentation achieved, the copy numberof an internal fragment of the amplicon was compared before and afterfragmentation, using quantitative PCR without UNG. The cycle delayobserved due to fragmentation is an indication of the degree offragmentation (and of ribonucleotide incorporation). Thus, increasedribonucleotide incorporation leads to an increased Ct delay. For thisamplification, the reaction mixture was composed of 100 mM Tricine pH8.3, 50 mM KOAc, 5% v/v glycerol, 2.5 mM Mg(OAc)₂, 20 nM GQDS, 0.5%DMSO, 0.1×SYBR Green I, 2.5% v/v enzyme storage buffer (50% v/vglycerol, 100 mM KCl, 20 mM Tris pH 8.0, 0.1 mM EDTA, 1 mM DTT, 0.5%Tween 20), 200 μM each dCTP, dGTP, and dATP, 360 μM dUTP, and 40 μMdTTP. This reaction mix was used to generate a 340 bp product from thefragmented and unfragmented amplicons, diluting these templates afurther 10,000-fold from the dilution used for fragmentation. The primersequences were used at 200 nM each, wherein each primer comprised a2′-amino-C.

Reactions were run in 384-well plates, 20 μl per reaction in a kineticthermocycler. The thermocycling parameters were: 50° C. for 15 seconds;92° C. for 1 minute; then 46 cycles of: 92° C. for 15 seconds; followedby an extension step of 62° C. for 1 minute. Threshold Cts weredetermined and corresponding fragmented and unfragmented Cts werecompared, generating a delta Ct for each enzyme/rNTP condition tested.These are shown in FIGS. 16A and 16B. More specifically, FIGS. 16A and16B illustrate, e.g., that an increase in the degree of fragmentationcan be achieved by the mutant enzymes when using either rCTP orbiotinylated rCTP, because they are able to produce amplicon with ahigher level of ribonucleotide incorporation than the correspondingparental enzymes.

Example VII: Pyrophosphorolysis Activated Polymerization

The abilities of G46E L329A E678G CS5 DNA polymerase and G46E L329A D640S671F E678G CS5 DNA polymerase to perform pyrophosphorolysis activatedpolymerization (“PAP”) were compared. The reaction buffer was comprisedof 100 mM Tricine pH 8.0, 2.5-50 mM G46E L329A E678G CS5 DNA polymeraseor 2.5-50 mM G46E L329A D640G S671F E678G CS5 DNA polymerase, 50 nMKOAc, 10% v/v glycerol, 0.04 U/μl UNG, 4 mM Mg(OAc)₂, 1% DMSO, 0.2×SYBRGreen I, 2.5% v/v enzyme storage buffer (50% v/v glycerol, 100 mM KCl,20 mM Tris pH 8.0, 0.1 mM EDTA, 1 mM DTT, 0.5% Tween 20), 0.2 mM eachdATP, dCTP, and dGTP, and 0.4 mM dUTP, and 100 μM pyrophosphate. M13template and enzyme were cross-titrated. M13 concentrations used were 0,10⁴, 10⁵, and 10⁶ copies per 20 μl reaction. Enzyme concentrations usedwere 2.5 nM, 5 nM, 10 nM, 15 nM, 20 nM, 25 nM, 35 nM, and 50 nM.Reactions were set up in triplicate in a 384-well thermocycler, usingthe following cycling parameters: 50° C. for 2 minutes; 90° C. for 1minute; then 46 cycles of: 90° C. for 15 seconds followed by anextension temperature of 62° C. for 60 seconds.

One of the primers comprised a 2′-amino-C at the 3′-end and the otherprimer comprised a 2′-PO₄-A (i.e., a 2′-terminator nucleotide) at the3′-end. These primers, added to the reaction mix at 0.1 μM each, willresult in a 348 bp product from M13 template. However, the 2′-PO₄-Aresidue at the 3′-end of the second primer effectively acts as aterminator. In order to serve as a primer, it must be activated bypyrophosphorolytic removal of the terminal residue.

Fluorescence data was analyzed to determine elbow values (C(t))(emergence of fluorescence over baseline). C(t) values for G46E L329AE678G CS5 DNA polymerase are shown in FIG. 17. C(t) values for G46EL329A D640G S671F E678G CS5 DNA polymerase are shown in FIG. 18.Further, FIGS. 17 and 18 show, e.g., that using the mutant enzymeresults in more efficient PAP-PCR at lower enzyme concentrations thanthe corresponding non-mutant or parental enzyme. By gel analysis, theamplicon in the no template reactions in this example was specificproduct that likely arose from environmental M13.

Example VIII: Effect of Selected Mutations on the Extension Rate ofThermus Sp. Z05 DNA Polymerase

Several of the mutations isolated by the screen described in Example Iwere transferred to Thermus sp. Z05 DNA polymerase (see, e.g., U.S. Pat.No. 5,455,170, entitled “MUTATED THERMOSTABLE NUCLEIC ACID POLYMERASEENZYME FROM THERMUS SPECIES Z05” issued Oct. 3, 1995 to Abramson et al.and U.S. Pat. No. 5,674,738, entitled “DNA ENCODING THERMOSTABLE NUCLEICACID POLYMERASE ENZYME FROM THERMUS SPECIES Z05” issued Oct. 7, 1997 toAbramson et al., which are both incorporated by reference). First, theamino acid position corresponding to the mutations were determined byusing the alignment shown in FIG. 1. These were named as follows:“Q”=T541R; “D”=D580G; and “S”=A610F. These mutations were introducedinto a plasmid encoding Z05 DNA polymerase by using the method known asoverlap extension PCR (see, e.g., Higuchi, R. in PCR Protocols: A Guideto Methods and Applications, ed. Innis, Gelfand, Sninsky and White,Academic Press, 1990, and Silver et. al., “Site-specific MutagenesisUsing the Polymerase Chain Reaction”, in “PCR Strategies”, ed. Innis,Gelfand, and Sninsky, Academic Press, 1995, which is incorporated byreference). In this method, two amplicons are first generated, oneupstream and one downstream of the site to be mutagenized, with themutation being introduced in one of the primers of each reaction. Theseamplification products are then combined and re-amplified using theoutside, non-mutagenic primers. The resulting amplicon includes theintroduced mutation and also is designed to span vector-uniquerestriction sites, which can then be used to clone the amplicon into thevector plasmid DNA. Diagnostic restriction sites may also be introducedinto the mutagenic primers as needed, in order to facilitate selectionof the desired mutation from the resulting clones, which may include amixture of mutants and wild-type clones. This procedure may introduceundesired mutations caused by low fidelity PCR, and hence it isnecessary to sequence the resulting clones to confirm that only thedesired mutations were created. Once the mutations were confirmed, theywere combined with each other or with the previously isolated E683Rmutation (ES112) (see, U.S. Pat. Appl. No. 20020012970, entitled “Hightemperature reverse transcription using mutant DNA polymerases” filedMar. 30, 2001 by Smith et al., which is incorporated by reference) byrestriction fragment swaps, as described previously.

Expression plasmids created in this way were used to make purifiedprotein of the various mutants, as described earlier in Example I. Thenucleic acid extension rate of the various mutants was then determined.The reaction mixture contained 25 mM Tricine pH 8.3, 100 mM KOAc, 3 mMMgCl₂, 2.5% v/v Storage Buffer (50% v/v glycerol, 100 mM KCl, 20 mM TrispH 8.0, 0.1 mM EDTA, 1 mM DTT, 0.5% Tween 20), 1% DMSO, 1×SYBR Green I,0.5 nM primed M13, and 5 nM enzyme. To this, nucleotides were added to afinal concentration of 0.1 mM dGTP, 0.1 mM dTTP, and 0.1 mM dCTP, and0.1 mM dATP. Parallel reactions containing no nucleotides were also setup. All reactions were run in quadruplicate in 20 μl volume in 384 wellthermocycler plates. The extension of primed M13 template was monitoredby fluorescence in a kinetic thermocycler set at 64° C., taking readingsevery 10 seconds. Identical reactions were averaged and the parallelminus nucleotide reactions subtracted. Extension rate (see, FIG. 19) wasestimated by linear regression analysis of the resulting data. This dataindicates, e.g., that in some cases the mutations described herein alsohave beneficial effects in the context of a non-chimeric Thermus DNApolymerase.

Example IX: HIV DNA Template Titrations

PAP-related HIV DNA template titrations were performed with and withoutthe presence of genomic DNA. FIG. 20 is a photograph of a gel that showsthe detection of the PCR products under the varied reaction conditionsutilized in this analysis. This data illustrates, e.g., the improvedamplification specificity and sensitivity that can be achieved using theblocked primers relative to reactions not using those primers.

More specifically, the reactions were performed using an ABI 5700Sequence Detection System with the following temperature profile:

50° C. 2 minutes

93° C. 1 minutes

93° C., 15 seconds→52° C., 4 minutes×4 cycles

90° C., 15 seconds→55° C., 4 minutes×56 cycles

The following reaction conditions were common to all reactions:

Master Mix Components conc. Tricine (pH 8.0) 100 mM dATP 200 μM dCTP 200μM dGTP 200 μM dTTP 30 μM dUTP 300 μM Primer 3 or Primer 1 200 nM Primer4 or Primer 2 200 nM KOAc 110 mM SYBR Green I 0.2X NaPPi 225 μM Mg(OAc)₂2.5 mM Tth Storage Buffer (0.2% Tween) 6% v/v GLQDSE CS5 DNA polymerase10 nM

Note, that “GLQDSE CS5 DNA polymerase” refers to a G46E L329A Q601RD640G S671F E678G CS5 DNA polymerase. Note further, that the “TthStorage Buffer” included 0.2% Tween 20, 20 mM Tris pH8.0, 0.1 mM EDTA,100 mM KCl, 1 mM DTT, and 50% v/v glycerol. In addition, each reactionvolume was brought to 50 μl with diethylpyrocarbonate (DEPC) treatedwater.

The varied reaction components included unblocked primers (see, thereactions denoted “unblocked primers” in FIG. 20) and primers blockedwith a 2′-Phosphate-U (i.e., a 2′-terminator nucleotide comprising aphosphate group at the 2′ position) (see, the reactions denoted “blockedprimers” in FIG. 20). The reactions also either included (see, thereactions denoted “25 ng Genomic DNA” in FIG. 20) or lacked (see, thereactions denoted “Clean Target” in FIG. 20) 25 ng of human genomic DNAadded to the mixtures. As further shown in FIG. 20, the reactions alsoincluded 10⁵, 10⁴, 10³, 10², or 10′ copies of linearized plasmid DNA,which included the target nucleic acid, diluted in 1 μl HIV SpecimenDiluent (10 mM Tris, 0.1 mM EDTA, 20 μg/mL Poly A, and 0.09% NaN₃) or 1μl HIV Specimen Diluent in “Neg” reactions. The indicated primer pairsamplified a 170 base pair product from the plasmid DNA.

Example X: Amplification of Mutant K-Ras Plasmid Template in aBackground of Wild-Type K-Ras Plasmid Template

Amplifications involving various copy numbers of mutant K-Ras plasmidtemplate in a background of wild-type K-Ras plasmid template andcomparing blocked and unblocked primers were performed. FIG. 21 is agraph that shows threshold cycle (C_(T)) values (y-axis) observed forthe various mutant K-Ras plasmid template copy numbers (x-axis) utilizedin these reactions. FIG. 21 further illustrates, e.g., the improveddiscrimination that can be achieved using the blocked primers describedherein.

The reactions were performed using an ABI 5700 Sequence Detection Systemwith the following temperature profile:

50° C. 2 minutes

93° C. 1 minute

92° C., 15 seconds→65° C., 2 minutes×60 cycles

The following reaction conditions were common to all reactions:

Master Mix Components conc. Tricine (pH 8.0) 100 mM dATP 200 μM dCTP 200μM dGTP 200 μM dTTP 30 μM dUTP 300 μM Primer 7 or Primer 5 200 nM Primer8 or Primer 6 200 nM SYBR Green I 0.1X NaPPi 225 μM Mg(OAc)₂ 2.5 mM Ung2U Tth Storage Buffer (0.2% Tween) 6% v/v GDSE CS5 DNA polymerase 5 nMLinearized Wild-Type Plasmid DNA 10⁶ copiesNote, that “GDSE CS5 DNA polymerase” refers to a G46E D640G S671F E678GCS5 DNA polymerase. In addition, each reaction volume was brought to 50μl with DEPC treated water.

The varied reaction components included unblocked primers (see, thereactions denoted “unblocked” in FIG. 21) and primers blocked with a2′-Phosphate-C or a 2′-Phosphate-A (i.e., 2′-terminator nucleotidescomprising phosphate groups at 2′ positions). In addition, 10⁶, 10⁵,10⁴, 10³, 10², 10¹ or 0 copies (NTC reactions) (10e6c, 10e5c, 10e4c,10e3c, 10e2c, 10e1c, and NTC, respectively, in FIG. 21) of linearizedmutant K-Ras plasmid DNA were added to the reactions. The relevantsubsequences of the mutant plasmid DNA were perfectly matched to boththe blocked and unblocked primer sets. Further, the mutant K-Ras plasmidDNA was diluted in 1 μl HIV Specimen Diluent (see, above) or 1 μl HIVSpecimen Diluent (see, above) in “NTC” reactions. Additionally, 10⁶copies of linearized wild-type K-Ras plasmid DNA were present in allreactions. The wild-type K-Ras plasmid DNA was identical in sequence tomutant plasmid DNA except that it creates a C:C mismatch with theultimate 3′ base (dC) in primers 5 and 7. Both blocked and unblockedprimer pairs created a 92 base pair amplicon on the mutant linearizedplasmid template.

Example XI: Amplification of K-Ras Plasmid Template with Various Enzymesat Varied Concentrations

Amplifications involving K-Ras plasmid template with various enzymes atvaried concentrations were performed. FIG. 22 is a graph that showsthreshold cycle (C_(T)) values (y-axis) observed for the various enzymesand concentrations (x-axis) utilized in these reactions. These datashow, e.g., the improved PAP amplification efficiencies that can beachieved using certain enzymes described herein.

The reactions were performed using an ABI 5700 Sequence Detection Systemwith the following temperature profile:

50° C. 2 minutes

93° C. 1 minute

92° C., 15 seconds→60° C., 2 minutes×60 cycles

The following reaction conditions were common to all reactions:

Master Mix Components conc. Tricine (pH 8.0) 100 mM dATP 200 μM dCTP 200μM dGTP 200 μM dTTP 30 μM dUTP 300 μM Primer 9 200 nM Primer 10 200 nMSYBR Green I 0.1X NaPPi 225 μM Mg(OAc)₂ 2.5 mM Ung 2U Tth Storage Buffer(0.2% Tween) 6% v/v Linearized K-Ras Plasmid DNA 10⁴ copies

The reaction components included primers blocked with a 2′-Phosphate-Uor a 2′-Phosphate-A (i.e., 2′-terminator nucleotides comprisingphosphate groups at 2′ positions). The primer pairs created a 92 basepair amplicon on the linearized K-Ras plasmid template. In addition,each reaction volume was brought to 50 μl with diethylpyrocarbonate(DEPC) treated water.

The polymerase concentration and KOAc concentrations were optimized foreach individual polymerase as follows:

KOAc Polymerase Polymerase Conc. (nM) (mM) GLQDSE 5, 10, 15, 20, 30, or40 nM 110 GLDSE 5, 10, 15, 20, 30, or 40 nM 25 GLE 5, 10, 15, 20, 30, or40 nM 25Note, that “GLQDSE” refers to a G46E L329A Q601R D640G S671F E678G CS5DNA polymerase, “GLDSE” refers to a G46E L329A D640G S671F E678G CS5 DNApolymerase, and “GLE” refers to a G46E E678G CS5 DNA polymerase.

Example XII: Hepatitis C Virus (HCV) RNA to cDNA Reverse Transcription(RT) Comparing Unblocked and Blocked RT Primers

The extension of an unblocked HCV RT primer was compared to theextension of a blocked primer on an HCV RNA template in reversetranscription reactions. These RT comparisons were performed usingvarious polymerases. To illustrate, FIG. 23 is a graph that showsthreshold cycle (Ct) values (y-axis) observed for the various enzymes(x-axis) utilized in these reactions in which the cDNA was measuredusing real-time PCR involving 5′-nuclease probes.

The following reaction conditions were common to all RT reactions:

RT Mix Component Concentration Tricine pH 8.0 100 mM KOAc 100 mM DMSO 4%(v/v) Primer 1 or 2 200 nM dATP 200 μM dCTP 200 μM dGTP 200 μM dTTP 30μM dUTP 300 μM UNG 0.2 Unit Mn(OAc)₂ 1 mM PPi 175 uM

The varied reaction components included a 3′-OH unblocked primer (see,the reactions denoted “3′ OH Primer (Unblocked)” in FIG. 23) and aprimer blocked with a 2′-Phosphate-A or a 2′-monophosphate-3′-hydroxyladenosine nucleotide (i.e., 2′ terminator nucleotide comprising aphosphate group at the 2′ position) (see, the reactions denoted “2′ PO4(Blocked)” in FIG. 23). Further, the following polymerase conditionswere compared in the cDNA reactions (see, FIG. 23):

Z05 DNA polymerase (13 nM)

GLQDSE CS5 DNA polymerase (100 nM) combined with GLQDS CS5 DNApolymerase (25 nM)

GLQDSE CS5 DNA polymerase (50 nM) combined with GLQDS CS5 DNA polymerase(50 nM

where “GLQDSE CS5 DNA polymerase” refers to a G46E L329A Q601R D640GS671F E678G CS5 DNA polymerase and “GLQDS CS5 DNA polymerase” refers toa G46E L329A Q601R D640G S671F CS5 DNA polymerase. In addition, eachreaction was brought to 20 μl with diethylpyrocarbonate (DEPC) treatedwater.

The RT reactions were incubated at 60° C. for 60 minutes in an ABI 9600Thermal Cycler. After the RT incubation, RT reactions were diluted100-fold in DEPC treated water. The presence of cDNA was confirmed andquantitated by 5′ nuclease probe-based real-time HCV PCR reactionsdesigned to specifically measure the HCV cDNA products of the RTreactions. These reactions were performed using an ABI Prism 7700Sequence Detector with the following temperature profile:

50° C. 2 minutes

95° C. 15 seconds→60° C. 1 minutes×50 cycles.

Example XIII: Bidirectional Pap for BRAF Mutation Detection

FIG. 24 shows PCR growth curves of BRAF oncogene amplifications thatwere generated when bidirectional PAP was performed. The x-axis showsnormalized, accumulated fluorescence and the y-axis shows cycles of PAPPCR amplification. More specifically, these data were produced whenmutation-specific amplification of the T→A mutation responsible for theV599E codon change in the BRAF oncogene (see, Brose et al. (2002) CancerRes 62:6997-7000, which is incorporated by reference) was performedusing 2′-terminator blocked primers that overlap at their 3′-terminalnucleotide at the precise position of the mutation. When primersspecific to wild-type sequence were reacted to wild-type target ormutant target, only wild-type target was detected. Conversely, whenprimers specific to mutant sequence were reacted to wild-type target ormutant target, only mutant target was detected.

The following reaction conditions were common to all RT reactions:

Component Concentration Tricine pH 8.0 100 mM KOAc 100 mM Glycerol 3.5%v/v Primer F5W or F5M 200 nM Primer R5W or R5M 200 nM dATP 200 μM dCTP200 μM dGTP 200 μM dTTP 30 μM dUTP 300 μM UNG 1 Unit PPi 175 uM GLQDSE15 nM SYBR I/carboxyrhodamine 1/100,000 (0.1x) Mg(OAc)₂ 3.0 mMwhere “GLQDSE” refers to a G46E L329A Q601R D640G S671F E678G CS5 DNApolymerase.

The varied reaction components included the wild-type BRAF primersblocked with a 2′-Phosphate-A; a 2′-monophosphate-3′-hydroxyl adenosinenucleotide; a 2′-Phosphate-U; or a 2′-monophosphate-3′-hydroxyl uridinenucleotide (i.e., 2′ terminator nucleotides comprising a phosphate groupat the 2′ position) (labeled “F5W/R5W” in FIG. 24).

In addition, each reaction was brought to 50 μl with DEPC treated water.Wild-type reactions (labeled “WT” in FIG. 24) contained linearized DNAplasmid of the BRAF wild-type sequence and mutant reactions (labeled“MT” in FIG. 24) contained linearized DNA plasmid of the BRAF mutantsequence. Negative reactions (labeled “NEG” in FIG. 24) contained HIVspecimen diluent (10 mM Tris, 0.1 mM EDTA, 20 μg/mL Poly A, and 0.09%NaN₃) with no DNA. Combinations of the primers in PCR produced a 50 bpamplicon. Further, the reactions were performed using an ABI Prism 7700Sequence Detector with the following temperature profile:

50° C. 1 minutes

93° C. 1 minutes

90° C. 15 seconds

60° C. 150 seconds→×60 Cycles.

Example XIV: Detection of Fluorescent PAP Release Product

This prophetic example illustrates a real-time monitoring protocol thatinvolves PAP activation in which a blocked primer leads to theproduction of detectable signal as that primer is activated andextended.

Construction of a 3′ Terminated, Dual-Labeled Oligonucleotide Primer:

The primer QX below is a DNA oligonucleotide that includes a quenchingdye molecule, Black Hole Quencher® (BHQ) (Biosearch Technologies, Inc.)attached to the thirteenth nucleotide (A) from the 3′ terminus.

An oligonucleotide primer of the QX is mixed in solution with acomplimentary oligonucleotide R1 (see, below) such that they form ahybrid duplex. This duplex is further mixed with the reagents in theTable 10 provided below which notably include a fluorescein-labeleddeoxyriboadenine tetraphosphate (i.e., a fluorescein-labeled2′-terminator nucleotide) and DNA polymerase capable of incorporatingsuch labeled tetraphosphate. See, U.S. patent application Ser. No.10/879,494, entitled “SYNTHESIS AND COMPOSITIONS OF 2′-TERMINATORNUCLEOTIDES”, filed Jun. 28, 2004 and Ser. No. 10/879,493, entitled“2′-TERMINATOR NUCLEOTIDE-RELATED METHODS AND SYSTEMS,” filed Jun. 28,2004, which are both incorporated by reference. Incubation of themixture at a temperature of 60° C. for, e.g., one hour could causes the3′ terminus of the sequence QX to be extended one nucleotide in atemplate directed manner, resulting in at least a portion of the QXoligonucleotides being extended at their 3′ ends with thefluorescein-labeled deoxyriboadenine 2′-phosphate nucleotides,represented below as Primer QX^(FAM).

TABLE 10 Mix Component Concentration Tricine pH 8.3 50 mM KOAc 100 mMGlycerol 8% (w/v) Primer QX 10 μM Oligonucleotide R1 15 μM FluoresceindA4P 15 μM G46E L329A E678G CS5 DNA 50 nM polymerase Mg(OAc)₂ 2.5 mM

The newly elongated Primer QX^(FAM) are purified from the mixture aboveusing any number of purification methods known to persons of skill inthe art. An example of such a method capable of purifying PrimerQX^(FAM) from the mixture is High Performance Liquid Chromatography(HPLC). HPLC purification parameters are selected such that thepreparation of Primer QX^(FAM) is substantially free of non-extendedPrimer QX and fluorescein-labeled adenine tetraphosphates. Dual HPLC(Reverse Phase and Anion Exchange HPLC) is known as a method forpurifying such molecules.

Once purified, molecules such as Primer QX^(FAM) which contain a BHQquenching molecule and a fluorescein molecule on the sameoligonucleotide generally exhibit a suppressed fluorescein signal due toenergy absorbance by the BHQ2 “quencher” molecule.

Optionally, Primer QX^(FAM) is synthesized chemically as described in,e.g., U.S. Patent Publication No. 2007/0219361.

The sequences referred to in this example are as follows:

(SEQ ID NO: 73) Primer QX 5′-GCAAGCACCCTATCA^(Q)GGCAGTACCACA-3′(Where Q represents the presence of a BHQ molecule)

(SEQ ID NO: 74) R1 3′-PCGTTCGTGGGATAGTCCGTCATGGTGTT-5′(Where P represents 3′ phosphate)

(SEQ ID NO: 75) Primer QXFAM 5′-GCAAGCACCCTATCA^(Q)GGCAGTACCACA^(F)-3′(Where Q represents the presence of a BHQ molecule, and F represents afluorescein-labeled 2′ phosphate adenine)

(SEQ ID NO: 76) Primer HC2 5′-GCAGAAAGCGTCTAGCCATGGCTTA-3′.

Use of the Primer in PCR.

A Primer QX^(FAM) is combined with the reagents in Table 11.

TABLE 11 Component Concentration Tricine pH 8.0 100 mM KOAc 100 mMGlycerol 3.5% (v/v) DMSO 5% (v/v) Primer QX^(FAM) 150 nM Primer HC2 150nM dATP 200 μM dCTP 200 μM dGTP 200 μM dTTP 30 μM dUTP 300 μM UNG 1 UnitPPi 175 μM GLQDSE 15 nM Target sequence 10⁶ copies Mg(OAc)₂ 3.0 mM

In addition each reaction is brought to 50 μl with DEPC treated water.Some reactions contain a target sequence which serves as a substrate forPCR amplification, while others contain no target. For example, thetarget can be a DNA sequence identical to the 5′UTR region of the HCVgenome. Combinations of these primers in PCR are expected to produce anapproximately 244 bp amplicon.

The reactions can be performed using an ABI Prism 7700 Sequence Detectorwith the following temperature profile:

50° C. 1 minute

93° C. 1 minute

90° C. 15 seconds

60° C. 150″→×60 Cycles

For such a PCR to progress, PAP activation of the fluorescein-terminatedPrimer QX^(FAM) is necessary, and would result in the removal of thefluorescein-labeled deoxyadenine tetraphosphate molecule. Such a releaseis expected to result in an increase in fluorescent signal atapproximately 520 nm wavelength. With monitoring of signal atapproximately 520 nm wavelength as the PCR progresses, one would expectto observe an increase in fluorescence in those reactions containingtarget nucleic acid while observing no increased fluorescence inreactions that do not contain target.

Example XV: Effect D580K, D580L, D580R and D580T Mutations on theExtension Rate Z05 DNA Polymerase

The effect of various substitutions at the D580 position on the nucleicacid extension rate of Z05 DNA polymerase was determined. First, themutations were created in Z05 DNA polymerase, utilizing the technique ofoverlap PCR, and the mutant enzymes purified and quantified, asdescribed previously. The extension rate on primed M13 (single-strandedDNA) template was determined, using both Mg⁺² and Mn⁺² as the metalco-factor, by monitoring the increase in SYBR Green I florescence, asdescribed in Example II above, and elsewhere. In this example, thereaction mixture contained 50 mM Tricine pH 8.3, 40 mM KOAc, 1 mMMn(OAc)₂ or 2.5 mM Mg(OAc)₂, 1.25% v/v Storage Buffer (50% v/v glycerol,100 mM KCl, 20 mM Tris pH 8.0, 0.1 mM EDTA, 1 mM DTT, 0.5% Tween 20), 1%DMSO, 0.6×SYBR Green I, 1.0 nM primed M13, and or 5 nM enzyme. To this,nucleotides were added to a final concentration of 0.2 mM dGTP, 0.2 mMdTTP, and 0.2 mM dCTP, and 0.2 mM dATP. Parallel reactions containing nonucleotides were also set up. All reactions were run in quadruplicate in20 μl volume in 384 well thermocycler plates. The extension of primedM13 template was monitored by fluorescence in a kinetic thermocycler setat 64° C., taking readings every 15 seconds. Replicate identicalreactions were averaged and the parallel minus nucleotide reactionssubtracted. Extension rate was estimated by linear regression analysisof the resulting data. Results are shown in Table 12 below:

TABLE 12 Extension Rate of D580X Mutants of Z05. (Based on Change inFluorescence, arbitrary units) Z05- Z05- Z05- Z05- Z05 Z05-D 580K 580L580R 580T   1 mM Mn 35.27 119.81 185.25 87.31 171.56 153.46 2.5 mM Mg127.94 216.59 258.69 176.59 237.58 238.33

The data indicate that all 5 amino acid substitutions at position 580 ofZ05 DNA polymerase result in faster extension rate under the conditionstested.

Example XVI Use of Various Mutant Z05 DNA Polymerases in RT-PCR

Mn²⁺-Based RT:

The mutations D580G, D580K, and D580R were evaluated for their effect onRT-PCR efficiency in the presence of Mn⁺². The reactions all containedthe following components: 55 mM Tricine pH 8.3, 4% v/v glycerol, 5% v/vDMSO, 110 mM KOAc, 2.7 mM Mn(OAc)₂, 3.6% v/v Storage Buffer (50% v/vglycerol, 100 mM KCl, 20 mM Tris pH 8.0, 0.1 mM EDTA, 1 mM DTT, 0.2%Tween 20), 0.04 units/μl UNG, 0.45 mM each dATP, dCTP, dUTP, dGTP; 750nM of each primer, wherein each primer comprised a t-butyl benzyl dA atthe 3′-end; and 150 nM of a TaqMan probe, labeled with a cyclohexyl-FAM,a black hole quencher (BHQ-2), a 3′-Phosphate. Together, the two primersgenerate a 241 bp amplicon on the HCV-1B transcript. 10⁵ copies of RNAtranscript HCV-1B was added to each 100 μl reaction.

Parallel reactions with no transcript were also set up. Each enzyme wasadded to a final concentration of 27 nM. Reactions were run in a RocheLC480 kinetic thermocycler. The thermocycling conditions were: 5 minutesat 50° C. (“UNG” step); 2, 5, or 30 minutes at 66° C. (“RT” step); 2cycles of 95° C. for 15 seconds followed by 58° C. for 50 seconds; and50 cycles of 91° C. for 15 seconds followed by 58° C. for 50 seconds.

Table 13 shows the Ct values obtained from the FAM signal increase dueto cleavage of the TaqMan probe:

TABLE 13 Z05 Z05 Z05 RT time Z05 D580G D580R D580K 30 min. RT  23.6 22.823.2 23.1 5 min. RT 27.9 23.4 23.3 23.2 2 min. RT 31.3 23.5 23.3 23.1

The results indicate these three mutations at position D580 allow for amuch shorter RT time while maintaining equivalent RT efficiency.

Mg²⁺-Based RT:

The mutations D580G and D580K, were compared to ES112 (Z05 E683R) fortheir ability to perform RT-PCR in the presence of Mg⁺². The parentalenzyme, Z05 DNA polymerase, is known to perform Mg⁺²-based RT-PCR withgreatly delayed Ct values relative to ES112, and was not re-tested inthis study. The conditions used were identical to those describedimmediately above, except that the KOAc was changed to 50 mM, theMn(OAc)₂ was replaced with 2 mM Mg(OAC)₂, and the enzyme concentrationwas reduced to 10 nM. Thermocycling conditions were identical, exceptthat only the 30 minute RT time was tested.

Table 14 shows the Ct values obtained from the FAM signal increase dueto cleavage of the TaqMan probe:

TABLE 14 Z05 Z05 RT time ES112 D580G D580K 30 min. RT 30.9 31.5 25.1

The results indicate the D580G mutant performs Mg⁺²-based RT PCR withroughly the same efficiency as does ES112, and that the D580K mutantresults in significantly a earlier Ct value, indicative of a much higherRT efficiency under these conditions.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference in theirentirety for all purposes.

1. A DNA polymerase comprising at least one motif in the polymerasedomain selected from the group consisting of: a)X_(a1)-X_(a2)-X_(a3)-X_(a4)-R-X_(a6)-X_(a7)-X_(a8)-K-L-X_(a11)-X_(a12)-T-Y-X_(a15)-X_(a16)(SEQ ID NO:1); wherein X_(a1) is I or L; X_(a2) is L or Q; X_(a3) is Q,H or E; X_(a4) is Y, H or F; X_(a6) is E, Q or K; X_(a7) is I, L or Y;X_(a8) is an amino acid other than Q, T, M, G or L; X_(a11) is K or Q;X_(a12) is S or N; X_(a15) is I or V; and X_(a16) is E or D; b)T-G-R-L-S—S-X_(b7)-X_(b8)-P-N-L-Q-N (SEQ ID NO:2); wherein X_(b7) is Sor T; and X_(b8) is an amino acid other than D, E or N; and c)X_(c1)-X_(c2)-X_(c3)-X_(c4)-X_(c5)-X_(c6)-X_(c7)-D-Y-S-Q-I-E-L-R (SEQ IDNO:3); wherein X_(c1) is G, N, or D; X_(c2) is W or H; X_(c3) is W, A,L, or V; X_(c4) is an amino acid other than I or L; X_(c5) is V, F or L;X_(c6) is an amino acid other than S, A, V, or G; and X_(c7) is A or Lwherein the polymerase has an improved nucleic acid extension rateand/or an improved reverse transcription efficiency relative to anotherwise identical DNA polymerase wherein X_(a8) is an amino acidselected from Q, T, M, G or L; X_(b8) is an amino acid selected from D,E or N; and X_(c6) is an amino acid selected from S, A, V, or G.
 2. TheDNA polymerase of claim 1, wherein the polymerase comprises motifs b)and c), and X_(b8) is an amino acid other than D, E or N, and X_(c6) isan amino acid other than S, A, V, or G.
 3. The DNA polymerase of claim1, wherein the polymerase comprises motifs a), b) and c), and the aminoacid at position X_(a8) is R; the amino acid at position X_(b8) is G;the amino acid at position X_(c4) is F; and the amino acid at positionX_(c6) is F.
 4. The DNA polymerase of claim 1, wherein the DNApolymerase has at least 90% sequence identity to a polymerase selectedfrom the group consisting of: (a) a CS5 DNA polymerase (SEQ ID NO: 18);(b) a CS6 DNA polymerase (SEQ ID NO: 19); (c) a Thermotoga maritima DNApolymerase (SEQ ID NO: 77); (d) a Thermus aquaticus DNA polymerase (SEQID NO: 78); (e) a Thermus thermophilus DNA polymerase (SEQ ID NO: 79);(f) a Thermus flavus DNA polymerase (SEQ ID NO: 80); (g) a Thermusfiliformis DNA polymerase; (h) a Thermus sp. sps17 DNA polymerase (SEQID NO: 81); (i) a Thermus sp. Z05 DNA polymerase (SEQ ID NO: 82); (j) aThermotoga neopolitana DNA polymerase (SEQ ID NO: 85); (k) a Thermosiphoafricanus DNA polymerase (SEQ ID NO: 83); (l) a Thermus caldophilus DNApolymerase (SEQ ID NO: 84).
 5. The DNA polymerase of claim 1, whereinthe DNA polymerase is a Z05 DNA polymerase, and the amino acid atposition X_(b8) is selected from the group consisting of G, T, R, K andL.
 6. (canceled)
 7. (canceled)
 8. A recombinant nucleic acid encodingthe DNA polymerase according to claim
 1. 9. An expression vectorcomprising the recombinant nucleic acid of claim
 8. 10. A host cellcomprising the expression vector of claim
 9. 11. A method of producing aDNA polymerase, said method comprising: culturing the host cell of claim10 under conditions suitable for expression of the nucleic acid encodingthe DNA polymerase.
 12. A method for conducting primer extension,comprising: contacting a DNA polymerase according to claim 1 with aprimer, a polynucleotide template, and free nucleotides under conditionssuitable for extension of the primer, thereby producing an extendedprimer.
 13. The method of claim 12, wherein the polynucleotide templateis an RNA.
 14. The method of claim 13, wherein the conditions suitablefor extension comprise Mg⁺⁺ and Mn⁺⁺.
 15. The method according to claim12, wherein the free nucleotides comprise unconventional nucleotides.16. The method of claim 12, which is a method for polynucleotideamplification comprising contacting the DNA polymerase with a primerpair, the polynucleotide template, and the free nucleotides underconditions suitable for amplification of the polynucleotide. 17.(canceled)
 18. (canceled)
 19. A kit for producing an extended primer,comprising: at least one container providing a DNA polymerase accordingto claim
 1. 20. The kit according to claim 19, further comprising one ormore additional containers selected from the group consisting of: (a) acontainer providing a primer hybridizable, under primer extensionconditions, to a predetermined polynucleotide template; (b) a containerproviding free nucleotides; and (c) a container providing a buffersuitable for primer extension.
 21. A reaction mixture comprising a DNApolymerase according to claim 1, at least one primer, a polynucleotidetemplate, and free nucleotides.
 22. The reaction mixture of claim 21,wherein the polynucleotide template is DNA.
 23. The reaction mixture ofclaim 21, wherein the polynucleotide template is RNA.
 24. (canceled) 25.The DNA polymerase of claim 1, wherein the DNA polymerase has at least95% sequence identity to the amino acid sequence selected from the groupconsisting of SEQ ID NO: 18; SEQ ID NO: 19; SEQ ID NO: 77; SEQ ID NO:78; SEQ ID NO: 79; SEQ ID NO: 80; SEQ ID NO: 81; SEQ ID NO: 82; SEQ IDNO: 83; SEQ ID NO: 84; and SEQ ID NO: 85.